Compounds for enzyme amplification assay

ABSTRACT

Novel biological assay method for determining the presence of a specific organic material by employing a modified enzyme for amplification. By employing receptors specific for one or a group of materials (hereinafter referred to as &#34;ligands&#34;) and binding an enzyme to the ligand or ligand counterfeit to provide an &#34;enzyme-bound-ligand&#34;, an extremely sensitive method is provided for assaying for ligands. The receptor when bound to the enzyme-bound-ligand substantially inhibits enzymatic activity, providing for different catalytic efficiencies of enzyme-bound-ligand and enzyme-bound-ligand combined with receptor.

CROSS REFERENCE TO RELATED APPLICATION

This application is a continuation, of application Ser. No. 481,022,filed June 20, 1974, now abandoned which is a division of applicationSer. No. 304,157, filed Nov. 6, 1972 now U.S. Pat. No. 3,852,157 whichis a Continuation-in-Part of Application Ser. No. 143,609, filed May 14,1971, now abandoned.

BACKGROUND OF THE INVENTION

1. Field of the Invention

There is a continually pressing need for rapid, accurate qualitative andquantitative determinations of biologically active substances atextremely low concentrations. The purpose of the determination can beextremely varied. Today, there is a wide need for determining thepresence of drugs or narcotics in body fluids, such as saliva, blood orurine. In addition, in medical diagnosis, it is frequently important toknow the presence of various substances which are synthesized naturallyby the body or ingested. These include hormones, both steroidal andpolypeptides, prostaglandins, toxins, as well as other materials whichmay be involved in body functions. Frequently, one is concerned withextremely small amounts and occassionally, with very small differencesin concentrations.

To meet these needs, a number of ways have been devised for analyzingfor trace amounts of materials. A common method is to use thin layerchromatography (TLC). By determining the flow factors and using specificreagents, the presence of certain materials can be detected; in manyinstances, the particular material can be isolated and identifiedquantitatively, for example, by mass spectroscopy or gas phasechromatography. However, thin layer chromatography has a number ofdeficiencies in being slow, requiring a high degree of proficiency inits being carried out, being subject to a wide range of interferingmaterials, and suffering from severe fluctuations in reliability.Therefore, the absence of satisfactory alternatives has resulted inintensive research efforts to determine improved methods of separationand identification.

An alternative to thin layer chromatography has been radioimmunoassay.Here, antibodies are employed for specific haptens or antigens. Aradioactive analog employing a radioactive atom of high flux is used andbound to the antigen. By mixing an antibody with solutions of the haptenor antigen and the radioactive hapten or antigen analog, the radioactiveanalog will be prevented from bonding to the antibody in an amountdirectly related to the concentration of the hapten or antigen in thesolution. By then separating the free radioactive analog from theantibody bound radioactive analog and determining the radioactivity ofthe separate components, one can determine the amount of hapten orantigen in the original solution.

The use of radioactive materials is not desirable for a variety ofreasons. First, radioactivity creates handling problems and undesirablehazards. Secondly, the preparation of such compounds involves similarhazards, greatly enhanced by the much larger amounts of radioactivematerials which are present. Because of their instability, theradioactive materials have only a short life. In addition, the use ofradioactive materials requires a license from the Atomic EnergyCommission, subjecting the licensee to review by the Commission as tothe maintenance of minimum operating standards. These standards maychange from time to time, so as to involve added expense andinconvenience to the licensee. Finally, the separation of the bound andunbound radioactive analog is difficult and subject to error. See, forexample, Abraham, Prelim. Comm., 29, 866 (1969).

Besides the aforementioned materials, assays at extremely lowconcentrations would be desirable for a variety of pesticides, such asinsecticides, bactericides, fungicides, etc., as well as other organicpollutants, both in the air and water. Organic pollutants may be assayedwhenever a receptor can be devised and the pollutant is inert to thereagents employed.

2. Description of the Prior Art

Use of radioimmunoassay is described in two articles by Murphy, J. Clin.Endocr. 27, 973 (1967); ibid., 28, 343 (1968). The use of peroxidase asa marker in an immunochemical determination of antigens and antibodiesis found in Stanislawski et al, C. R. Acad. Sci. Ser. D. 1970, 271 (16),1442-5. (C.A. 74 1144 B). See also, Nakane, et al, J. Histochem. andCytochem. 14, 929 (1967) and Avrameas, Int. Rev. of Cytology, 27, 349(1970). A general description of thin layer chromatography for assay maybe found in Stahl, Thin Layer Chromatography, Springer Verlag, New York,1969. See also, Peron, et al, Immunologic Methods in SteroidDetermination, Appleton, Century Crofts, New York, 1970.

Also of interest are publications by Van Weemen, et al, FEBS Letters 14,232 (1971), and Engvall, et al, Immunochemistry, 8, 871 (1971) concernedwith immunoassays employing enzymes. See also U.S. Pat. No. 3,654,090.See also, Cinader, Proceedings of the Second Meeting of the Foundationof European Biochemical Societies, Pergamon, Oxford, 1967, vol. IIchapter four.

SUMARY OF THE INVENTION

Detection of ligands is obtained at extremely low concentrations byusing specific receptor sites for the ligand and enzyme amplification ofligand displacement. By bonding a ligand or a ligand counterfeit to anenzyme while retaining enzymatic activity and then combining theenzyme-bound-ligand to a receptor for the ligand, the presence andamount of ligand in an unknown solution may be readily determined. Bycompetition for receptor sites between the enzyme-bound-ligand and thefree ligand, the two ligand moieties being added to the receptorsimultaneously or sequentially, the difference in enzymatic activityresulting from the presence or absence of ligand may be determined inaccordance with a particular analytical scheme. This difference will berelated to the amount of ligand present in the unknown solution.Enzymatic activity is easily determined in known ways by following thechange in concentration of an enzyme substrate or product of thesubstrate by standard techniques.

DESCRIPTION OF THE SPECIFIC EMBODIMENTS

This invention provides a method for detecting or assaying extremely lowconcentrations of a wide range of organic materials by relating thepresence of a particular unknown to enzymatic activity. An amplificationis obtained by having a large number of molecules formed or transformedas a result of the presence of one molecule. This amplification isachieved by bonding the compound to be assayed or a counterfeit of thecompound to an enzyme. This assemblage is referred to as anenzyme-bound-ligand. The particular molecule to be assayed is referredto as a ligand. The ligand analog will include either a ligand which ismodified by replacing a proton with a linking group to bond to theenzyme or a ligand counterfeit which is a ligand modified by other thansimple replacement of a proton to provide a linking site to the enzyme.The ligand and the enzyme-bound-ligand are both capable of binding in acompetitive fashion to specific receptor sites. It should also be notedthat other compounds of very similar structure may serve as ligandscapable of competing for these sites, e.g., morphine glucuronide andcodeine will compete with enzyme-bound-morphine for binding to certaintypes of morphine antibodies. In most instances, this is advantageous inpermitting one to assay for a class of physiologically closely relatedcompounds.

Various methods or protocols may be employed in assaying for a widevariety of ligands. Normally, the ligand, enzyme-bound-ligand andreceptor will be soluble in the medium employed. The substrate(s) forthe enzyme may or may not be soluble in the medium. In some situationsit may be desirable to provide a synthetic substrate which is notsoluble or employ an insoluble natural substrate.

In carrying out the assay, the enzyme-bound-ligand is combined with ahigh molecular weight receptor which results in inhibition of enzymaticactivity. When a ligand and enzyme-bound-ligand are introduced into asolution containing ligand receptor, the enzymatic activity of thesolution after the three substances are combined will be affected by theconcentration of the ligand present in the solution. That is, theenzyme-bound-ligand and the ligand will compete for the receptor sites.The number of enzyme-bound-ligand molecules not inhibited by thereceptor will be directly related to the number of ligand moleculespresent in the solution. One can achieve this in two ways: (1) either bycompetition, whereby the enzyme-bound-ligand and ligand are introducedto the receptor substantially simultaneously; or (2) theenzyme-bound-ligand or ligand may be first added to the receptor, andthe system allowed to come to equilibrium, and then the ligand added orenzyme-bound-ligand added respectively, in effect, to displace thematerial originally added from the receptor. Since the enzymaticactivity will be diminished or inhibited when the enzyme-bound-ligand isbound to the receptor, the enzymatic activity of the solution will bedirectly related to the amount of ligand present in the solution.

The assay can be carried out, either by considering the effect of ligandon the rate at which enzyme-bound-ligand binds to receptor or the effectof ligand on the equilibrium between the reagents: enzyme-bound-ligandand receptor. Where enzyme-bound-ligand and ligand are present withreceptor, one need not wait until equilibrium is achieved between thethree species. If one measures the enzymatic activity at a specific timeor interval of time from the time of combination of the three species,the enzymatic activity of the assay mixture will be a function of theeffect of the ligand on the rate of binding of the enzyme-bound-ligandto the receptor. By determining standards under the same conditions,including the same time interval, employing different concentrations ofligand, a smooth standard curve is obtained.

By measuring the effect of the ligand on rate of binding, rather thanthe effect on equilibrium, a shorter time interval between the time ofcombining the reagents and unknown suspected of containing the ligandand the time for the determination will be involved, as compared withwaiting until equilibrium is achieved. It is frequently found thatreproducible values can be obtained in from 0.1 to 5 minutes aftercombining the reagents and unknown. The rate of enzymatic activity isusually determined over a short time interval, e.g., 1 minute. The timeinterval can be the second, third, etc. minute from the time when thereagents and unknown were combined.

The concentrations of the reagents: the enzyme-bound-ligand and thereceptor, may be varied widely. Normally, the concentration of receptor(based on active sites) and enzyme-bound-ligand will be from about 10⁻⁴to 10⁻¹⁴ M, more usually from 10⁻⁶ to 10⁻¹² M. The lower limit for theconcentration of enzyme-bound-ligand is predicated on the minimum amountwhich can be detected. This will vary with different enzymes as well asdifferent detection systems.

The amount of receptor employed is normally calculated based on receptorsites and will vary with the concentration of enzyme-bound-ligand, theratio of ligand to enzyme in the enzyme-bound-ligand, and the affinityof the receptor for the ligand. Usually, there will be at least 1 activereceptor site per molecule of enzyme-bound-ligand and less than about 20active sites per molecule of ligand as enzyme-bound-ligand, butsite-ligand molecule ratios may be as high as 1,000 to 1, depending onthe type of assay and the affinity of the receptor. Preferably, theratio of receptor active sites to molecules of enzyme-bound-ligand willbe at least one, usually at least two, and the ratio of active sites tomolecules of ligand as enzyme-bound-ligand will be less than about 5to 1. The ratio will vary to a great degree depending on bindingconstants and the amount of ligand suspected of being present. Themethod of determining binding sites for the receptor will be discussedsubsequently in the experimental section.

The enzyme-bound-ligand will usually have molecules of ligand to enzymesubunit ratios on the average over the entire composition in the rangeof 0.01 - 100:1, frequently 0.02 - 50:1, and more frequently about0.04 - 25:1, wherein the number of ligands when the ligand is a proteinis expressed as the number of ligand molecules times the number of itscomponent polypeptide chains. For small ligands (less than about 10,000molecular weight), there will generally be at least one ligand, moreusually at least two ligands per enzyme, while with large ligands(greater than about 5,000 molecular weight) there will generally be atleast one enzyme per ligand. In the area of overlap, the ratio willdepend on the nature of the ligand, among other factors to be discussed.

The number of small ligands per enzyme will be affected to some degreeby the molecular weight of the enzyme. However, normally, the fewermolecules of ligand bound to an enzyme to achieve the desired degree ofinhibitability with receptor, the more sensitive the assay. Therefore,the number of small ligands per enzyme will usually not exceed 40, moreusually not exceed 30, and will not exceed 1 ligand per 2,000 molecularweight of enzyme on the average over the entire composition. Usually,the range of ligands will be 1 to 40, more usually 1 to 24, and withrandom substitution 2 to 20.

With large ligands, there will be on the average not more than oneenzyme per 2,000 molecular weight, usually not more than one enzyme per4,000 molecular weight, and more usually not more than one enzyme per6,000 molecular weight.

In some instances, a number of enzymes bind together in a stablearrangement to form a multienzyme complex. Because of the juxtapositionof the enzymes, a number of reaction may be carried out sequentially inan efficient manner, providing localized high concentrations ofreactants. Therefore, the ligand may be bound to a combination ofenzymes, whereby there will be a plurality of enzymes per ligand. If anumber of ligands were bound to the multienzyme complex, one could have1:1 mole ratio of enzymes to ligand, although, in fact, there would be aplurality of enzymes and ligands involved in a single aggregation. Thenumber of enzymes bound together, either as a multienzyme complex or byanother mechanism will rarely exceed 20, usually not exceed 10, andcommonly be in the range of 2 to 5 enzymes.

All other things being equal, the greater the number of enzymes perlarge ligand, the greater the sensitivity of the assay. However, theenzymes may interfere with receptor recognition, affect solubility andbe deleterious in other ways. Therefore, usually, the number of enzymesbonded to a large ligand will be such that there will be no more thanone enzyme polypeptide chain for every 2,000 molecular weight of theligand.

The concentration of receptor and enzyme will be related to the range ofconcentration of the ligand to be assayed. The solution to be assayedwill be used directly, unless a relatively high concentration of ligandis present. If a high concentration is present, the unknown solutionwill be diluted so as to provide a convenient concentration. However, inmany biological systems of interest, the amount of material beingassayed will be relatively small and dilution of the unknown substratewill usually not be required.

To illustrate the subject method, a soluble receptor is employed for aparticular ligand. For illustrative purposes, the ligand will beconsidered the hapten, morphine, and the receptor will be an antibodyspecific for morphine. It should be noted parenthetically, thatantibodies generally recognize molecular shape and distribution of polargroups in a ligand, although a portion of the ligand may besignificantly modified without preventing recognition. For example, bothmorphine ant its glucuronide can be bound to certain morphineantibodies.

An enzyme is first modified by bonding one or more morphine molecules tothe enzyme; a sufficient number of morphine groups are employed so thatgreater than about 20% inhibition, usually 50% inhibition, andpreferably, at least 70% inhibition is obtained when the maximum numberof ligands are conjugated to receptor. Complete inhibition is usuallyneither necessary or desirable. In many instances, all that is requiredis that there be a measurable difference between completely uninhibitedand maximally inhibited enzyme-bound-ligand which would allow for asemi-quantitative or quantitative determination of a ligand through adesired range of concentrations. Any convenient enzyme can be used thatwill catalyze the reaction of a substrate that can be easily detectedand for which a substrate is available which allows for inhibition ofthe enzyme when bound to receptor.

A solution is prepared of the antibody at the requisite concentration.Only a few microliters of solution are required. The antibody,maintained at a pH at which it is active in binding morphine, isintroduced into a solution of the enzyme-bound-morphine at the desiredconcentration. The reactivity of the combined antibody andenzyme-bound-morphine solution can be determined by taking an aliquot,adding it to its substrate under conditions where the enzyme is active,and determining the spectroscopic change as a function of time at aconstant temperature. The rate of this change will be the result thatshould be obtained when there is no morphine present in the unknownsolution.

Normally, the ligand and enzyme-bound-ligand reversably bind toreceptor, so that the order of addition of reagents is not crucial.

A second aliquot is taken and added to the unknown solution. The unknownsolution may contain the substrate and any other additives which arerequired for enzymatic activity. Alternatively, the unknown solution mayfirst be combined with the antibody-(enzyme-bound-morphine) complex,allowed to come to equilibrium and then mixed with the substrate. Ineither case the rate of change in the spectrum is determined. A variantof the above method is to add combined enzyme-bound-morphine and unknownsolution to the antibody and then add this solution to the substrate.Other obvious variations come readily to mind.

If all concentrations of reagents except morphine are kept constant andseveral standard solutions of morphine are employed, then one can relatethe change in the spectrum over specified periods of time to themorphine concentrations. Obviously, the standardized system can then beused to determine rpaidly, accurately, and efficiently the amount ofmorphine, or any other ligand in the unknown.

The manner of assaying for the enzyme can be widely varied depending onthe enzyme, and to some degree the ligand and the medium in which theligand is obtained. Conveniently, spectrophotometric measurements can beemployed, where absorption of a cofactor, a substrate or the product ofthe substrate absorbs light in the ultraviolet or visible region.However, in many instances other methods of determination may bepreferred. Such methods include fluorimetry, measuring luminenscence,ion specific electrodes, viscometry, electron spin resonancespectrometry, and metering pH, the name a few of the more popularmethods.

The assays will normally be carried out at moderate temperatures,usually in the range of from 10° to 50° C, and more usually in the rangeof about 15° to 40° C. The pH of the assay solutions will be in therange of about 5 to 10, usually about 6 to 9. Illustrative buffersinclude (trishydroxymethyl)methylamine salt, carbonate, borate andphosphate.

Whether oxygen is present or the assay is carried out in an inertatmosphere, will depend on the particular assay. Where oxygen may be aninterferant, an inert atmosphere will normally be employed. Normally,hydroxylic media will be employed, particularly aqueous media, sincethese are the media in which the enzyme is active. However 0 to 40volume percent of other liquids may also be present as co-solvents, suchas alcohols, esters, ketones, amides, etc. The particular choice of thecosolvent will depend on the other reagents present in the medium, theeffect on enzyme activity, and any desirable or undesirable interactionswith the substrate or products.

As already indicated, antibodies will frequently recognize a family ofcompounds, where the geometry and spatial distribution of polar groupsare similar. Frequently, by devising the haptenic structure and themethod of binding to the antigen when producing the antibodies, thespecificity of the antibody can be varied. In some instances, it may bedesirable to use two or more antibodies, usually not more than sixantibodies, so that the antibody reagent solution will be able to detectan entire group of compounds, e.g., morphine and barbiturates. This canbe particularly valuable for screening a sample to determine thepresence of any member of a group of compounds or determining whether aparticular class of compounds is present, e.g., drugs of abuse or sexhormones. When combinations of antibodies are used, it will usually benecessary to employ corresponding combinations of enzyme-bound-ligands.

Ligand

Turning now to a general consideration of the reagents, the firstreagent to be considered is the ligand. Any ligand may be employed forwhich an appropriate receptor may be found having satisfactoryspecificity for the ligand. The recent literature contains an increasingnumber of reports of receptors for an increasingly wide variety ofbiologically active materials. Compounds for which receptors can beprovided range from simple phenylalkylamines, e.g., amphetamine, to veryhigh molecular weight polymers, e.g., proteins.

Among ligands which are drugs, will be compounds which act as narcotics,hypnotics, sedatives, analgesics, antipyretics, anaesthetics,psychotogenic drugs, muscle relaxants, nervous system stimulants,anticholinesterase agents, parasympathomimetic agents, sympathomimeticagents, α-adrenergic blocking agents, antiadrenergic agents, ganglionicstimulating and blocking agents, neuromuscular agents, histamines,antihistamines, 5-hydroxy-tryptamine and antagonists, cardiovasculardrugs, antiarrhythmic drugs, antihypertensive agents, vasodilator drugs,diuretics, pesticides (fungicides, antihelminthics, insecticides,ectoparasiticides, etc.), antimalarial drugs, antibiotics,antimetabolites, hormones, vitamins, sugars, thyroid and antithyroiddrugs, corticosteroids, insulin, oral hypoglemic drugs, tumor cells,bacterial and viral proteins, toxins, blood proteins, and theirmetabolites.

(A drug is any chemical agent that affects living protoplasm. (Goodman &Gilman, The Pharmacological Basis of Therapeutics, 3rd Ed., Macmillan,New York (1965).) A narcotic is any agent that produces sleep as well asanalgesia.)

Included among such drugs and agents are alkaloids, steroids,polypeptides and proteins, prostaglandins, catecholamines, xanthines,arylalkylamines, heterocyclics, e.g., thiazines, piperazines, indoles,and thiazoles, amino acids, etc.

Other ligands of interest besides drugs are industrial pollutants,flavoring agents, food additives, e.g., preservatives, and foodcontaminants.

Broadly, the ligands will be organic compounds of from 100 to 100,000molecular weight, usually of from about 125 to 40,000 molecular weight,more usually 125 to 20,000 molecular weight. The ligand will usuallyhave from about 8 to 5,000 carbon atoms and from about 1 to 3,500heteroatoms.

A substantial portion of the ligands will be monomers or low orderpolymers, which will have molecular weights in the range of about 100 to2,000, more usually 125 to 1,000. Another significant portion of theligands will be polymers (compounds having a recurring group) which willhave molecular weights in the range of from about 750 to 100,000,usually from about 2,000 to 60,000, more usually 2,000 to 50,000. Forpolymers of varying molecular weight, weight average molecular weight isintended.

In some instances, high molecular weight materials will be of interest.For example, blood proteins will generally be in excess of 100,000molecular weight. In the case of lipoproteins, the molecular weight willbe in the range of 3 million to 20 million. The globulins, albumins andfibrinogens will be in the range of 100,000 to 1,000,000.

The ligands will normally be composed of carbon, hydrogen, nitrogen,oxygen, sulfur, phosphorous, halogen, and metals, primarily as theircations, such as the alkali and alkaline earth metals and the metals ofGroups IB, IIB, VIIB, and VIIIB, particularly the third row of theperiodic chart. Most usually, the ligands will be composed primarily ofcarbon, hydrogen, nitrogen, oxygen and sulfur.

Structurally, the ligands may be monomers or polymers, acyclic, mono orpolycyclic, having carbocyclic or heterocyclic rings. The ligands willhave a wide variety of functionalities, such as halo, oxocarbonyl,nonoxocarbonyl, amino, oxy (hydroxy, aryloxy, alyloxy and cycloalyloxy["alyl" intends a monovalent aliphatic radical]), thiooxy, dithio,hydrazo, and combinations thereof.

The ligands may be divided into three different categories, based ontheir biological relationship to the receptor. The first category isantigens, which when introduced into the bloodstream of a vertebrate,result in the formation of antibodies. The second category is haptens,which when bound to an antigenic carrier, and the hapten bound antigeniccarrier is introduced into the bloodstream of a vertebrate, elicitformation of antibodies specific for the hapten. The third category ofligands includes those which have naturally occurring receptors in aliving organism and the receptors can be isolated in a form specific forthe ligand.

Of course, biological substances which are native to one species andhave naturally occurring receptors in that species, may also be haptenswhen bonded to a protein and introduced into an animal of the same or adifferent species. Therefore, the classification is somewhat arbitraryin that the ligand may be an antigen as to one species, a hapten as toanother species, and may have naturally occurring receptors in a thirdspecies.

Antigens are for the most part protein or polysaccharide in nature andforeign to the animal into which they are injected.

The most important body of ligands for the purposes of the invention arethe haptens. "Substances which on injection do not give rise toantibodies, but which are able to react with antibodies specifically toproduce either precipitation or to inhibit precipitation have beentermed haptens. This definition has been used to include not only thesimple chemical substances which are determinants of specificity whenconjugated to protein, and which inhibit precipitation, but alsosubstances obtained from natural sources such as the pneumococcal typespecific polysaccharides and dextran which are not antigenic in therabbit on primary injection." Kabat, et al, ExperimentalImmunochemistry, Charles C. Thomas, Springfield, Illinois (1967). In thefollowing discussion the term hapten will be confined to groupsartificially introduced into antigenic carriers which promote theformation of antibodies to those groups.

The third group of ligands are those which have naturally occurringreceptors. The receptors may be proteins, nucleic acids, such asribonucleic acid (RNA) or deoxyribonucleic acid (DNA), or membranesassociated with cells. Illustrative ligands which have naturallyoccurring receptors are thyroxine, many steroids, such as the estrogens,cortisone, corticosterone, and estradiol; polypeptides such as insulinand angiotensin, as well as other naturally occurring biologicallyactive compounds. See Murphy, et al, J. Clin. Endocr., 24, 187 (1964);Murphy, ibid, 27, 973, (1967); ibid, 28, 343 (1968); BBA, 176, 626,(1969); McEwen, et al, Nature, 226, 263 (1970); Morgan, et al, Diabetes,(1966); Page, et al, J. Clin. Endocr., 28, 200, (1969).

The ligands may also be categorized by the chemical families which havebecome accepted in the literature. In some cases, included in the familyfor the purpose of this invention, will be those physiomimeticsubstances which are similar in structure to a part of the naturallyoccurring structure and either mimic or inhibit the physiologicalproperties of the natural substances. Also, groups of syntheticsubstances will be included, such as the barbiturates and amphetamines.In addition, any of these compounds may be modified for linking to theenzyme at a site that may cause all biological activity to be destroyed.Other structural modifications may be made for the ease of synthesis orcontrol of the characteristics of the antibody. These modified compoundsare referred to as ligand counterfeits.

A general category of ligands of particular interest are drugs andchemically altered compounds, as well as the metabolites of suchcompounds. The interest in assaying for drugs varies widely, fromdetermining whether individuals have been taking a specific illicitdrug, or have such drug in their possession, to determining what drughas been administered or the concentration of the drug in a specificbiological fluid.

The drugs are normally of from eight carbon atoms to 40 carbon atoms,usually of from 9 to 26 carbon atoms, and from 1 to 25, usually from 1to 10 heteroatoms, usually oxygen, nitrogen or sulfur. A large categoryof drugs have from one to two nitrogen atoms.

One class of drugs has the following basic functionality: ##STR1## wherethe lines intend a bond to a carbon atom, and wherein any of the carbonatoms and the nitrogen atom may be bonded to hydrogen, carbon or aheterofunctionality. Drugs which have this basic structure include theopiates such as morphine and heroin, meperidine, and methadone.

Another class of drugs are the epinephrine like drugs which have thefollowing basic functionality: ##STR2## where the lines intend a bond toa carbon atom and wherein any of the carbon atoms and the nitrogen atommay be bonded to hydrogen, carbon or a heterofunctionality. Drugs whichhave this basic structure include amphetamine, narceine, epinephrine,ephedrine and L-dopa.

The ligand analogs of drugs will usually have molecular weights in therange of 150 to 1,200 more usually in the range of 175 to 700.

Alkaloids

The first category is the alkaloids. Included in the category ofalkaloids, for the purpose of this invention, are those compounds whichare synthetically prepared to physiologically simulate the naturallyoccurring alkaloids. All of the naturally occurring alkaloids have anamine nitrogen as a heteroannular member. The synthetic alkaloids willnormally have a tertiary amine, which may or may not be a heteroannularmember. The alkaloids have a variety of functionalities present on themolecule, such as ethers, hydroxyls, esters, acetals, amines, isoxazole,olefins, all of which, depending on their particular position in themolecule, can be used as sites for bonding to the enzyme.

Opiates

The opiates are morphine alkaloids. All of these molecules have thefollowing functionality and minimum structures: ##STR3## wherein thefree valences are satisfied by a wide variety of groups, primarilycarbon and hydrogen.

The enzyme-bound-ligand analog of these compounds will for the most parthave the following minimum skeletal structure: ##STR4## wherein X is abond or a functionality such as imino, azo, oxy, thio, sulfonyl,oxocarbonyl, nonoxocarbonyl, or combinations thereof. Oxygen will be inthe ortho, meta or β position. A is an enzyme which is bonded to X atother than its reactive site and retains a substantial portion of itsnatural enzymatic activity. There will be m ligands bonded through X tothe enzyme A.

The enzyme-bound-morphine and its closely related analogs will have thefollowing formula: ##STR5## wherein:

any one of the W groups can be -X* or an H of any of the W groups may bereplaced by -X*, wherein X* is a bond or a linking group;

A* is an enzyme bonded at other than its reactive site, having a number(n) of ligands in the range of 1 to the molecular weight of A* dividedby 2,000, usually in the range of 2 to 40;

W¹ is hydrogen or hydrocarbon of from one to eight carbon atoms,particularly alkyl or alkenyl of from 1 to 4 carbon atoms,cycloalkylalkyl of from 4 to 6 carbon atoms, or aralkyl, e.g., methyl,allyl, 3-methylbut-2-enyl-1, cyclopropylmethyl and β-phenethyl;

W² is hydrogen;

W³ is hydrogen;

W⁴ is hydrogen or taken together with W³ a divalent radical of from 3 to6 carbon atoms and 0 to 2 oxygen atoms, forming a six memberedcarbocyclic ring with the carbon chain to which they are attached, e.g.,propylene-1,3,1-hydroxyprop-2-enylene-1,3,1-hydroxypropylene-1,3,1-acetoxypropylene-1,3, 1-acetoxyprop-2-enylene-1,3, 1-oxopropylene-1,3,1-oxoprop-2-enylene-1,3;

W⁵ is hydrogen or hydroxyl;

W⁶ is hydrogen, hydroxyl or taken together with W⁵ oxy (--O--);

W⁷ is hydrogen or methyl;

W⁸ is hydrogen, methyl or hydroxyl;

W⁹ is hydrogen, hydroxy, acyloxy of from 1 to 3 carbon atoms, e.g.,acetoxy, (unless otherwise indicated, acyl intends only nonoxocarbonyl),hydrocarbyloxy of from 1 to 3 carbon atoms, e.g., methoxy, ethoxy,2-(N-morpholino)ethoxy and glucuronyl; and

W^(9A) is hydrogen. (It is understood that in all the formulas, exceptwhen a minimum or skeletal structure is indicated, unsatisfied valencesare satisfied by hydrogen).

(Hydrocarbyl is an organic radical composed solely of hydrogen andcarbon and may be saturated or unsaturated, aliphatic, alicyclic,aromatic or combinations thereof).

By other than its reactive site, it is intended that the ligand is notbonded to the enzyme at a position which prevents the enzyme substrate,including necessary cofactors, from entering into the reaction catalyzedby the enzyme. It is understood, that with random substitution, theresulting product may include enzyme which has been deactivated byligand bonded at the reactive site, as well as enzyme which is activeand has ligand bonded at other than the reactive site.

The close morphine analogs will have the following formula: ##STR6##wherein:

any one of the W groups can be -X*;

-X*, A*, and n have been defined previously;

W^(1') is alkyl of from 1 to 3 carbon atoms, e.g., methyl;

W^(4') is hydrogen, hydroxy, oxo or acetoxy;

W^(5') is hydrogen or hydroxyl;

W^(6') is hydrogen, hydroxyl or taken together with W^(5') oxy (--O--);and

W^(9') is hydroxy, acetoxy, or alkoxy of from 1 to 3 carbon atoms;

Those preferred compounds having the basic morphine structure will havethe following formula: ##STR7## wherein:

one of W^(1") and W^(9") is -X**; when other than -X**;

W^(1") is methyl; and

W^(9") is hydrogen, methyl, acetyl or glucuronyl;

W^(4") is hydrogen or acetyl, usually hydrogen;

-X** is ##STR8## wherein Z is hydrocarbylene of from 1 to 7 carbonatoms, preferably aliphatic, having from 0 to 1 site of ethylenicunsaturation; and

-Z** is an enzyme, either specifically labelled with n' equal to 1 to 2ligands or randomly (random as to one or more particular availablereactive functionalities) labelled with n' equal to 2 to 30, moreusually 2 to 20, the enzyme retaining a substantial proportion of itsactivity. The enzyme will be of from about 10,000 to 300,000, frequentlyabout 10,000 to 150,000 molecular weight and is preferably anoxidoreductase, e.g., malate dehydrogenase, lactate dehydrogenase,glyoxylate reductase, or glucose 6-phosphate dehydrogenase, or aglycosidase, e.g., lysozyme or amylase.

Illustrative opiates which can be bound to an enzyme include morphine,heroin, hydromorphone, oxymorphone, metopon, codeine, hydrocodone,dihydrocodeine, dihydrohydroxycodeinone, pholcodine, dextromethorphan,phenazocine, and dionin and their metabolites.

Preferred compounds have W¹, or W⁹ as -X*-A* or have W³ and W⁴ takentogether to provide A*-X*-CHCH₂ CH₂ - or A*-X*-CH-CH═CH-.

Methadone

Another group of compounds having narcotic activity is methadone and itsanalogs, which for the most part have the following formula: ##STR9##wherein:

any one of the W groups can be X*;

X*, A*, and n have been defined previously;

p is 0 or 1, usually being the same in both instances;

q is 2 or 3;

W¹⁰ is hydrogen;

W¹¹ and W¹² are hydrogen, alkyl of from 1 to 3 carbon atoms, e.g.,methyl, or may be taken together to form a six-membered ring with thenitrogen atom to which they are attached, e.g., pentylene-1,5 and 3-oxaor 3-azapentylene-1,5;

W¹³ is hydrogen or methyl, only one W¹³ being methyl;

W¹⁴ is hydrogen;

W¹⁵ is hydrogen or hydroxyl;

W¹⁶ is hydrogen, acyloxy of from 1 to 3 carbon atoms, e.g., propionoxy,or hydroxy (when W¹⁵ and W¹⁶ are both hydroxy, the oxo group isintended); and

W¹⁷ is hydrogen or alkyl of from 1 to 3 carbon atoms, e.g., ethyl.

Illustrative compounds which can be linked to an enzyme are methadone,dextromoramide, dipipanone, phenadoxone, propoxyphene (Darvon) andacetylmethadol.

Metabolites of methadone and methadone analogs are also included. Amongthe metabolites for methadone is N-methyl2-ethyl-3,3-diphenyl-5-methylpyrroline.

Preferred compounds are when W¹¹ or W¹⁷ is -X*.

A narrower class of methadone and its analogs are of the formula:##STR10## wherein:

any one of the W groups can be -X*;

X*, A* and n have been defined previously;

W^(10') and W^(14') are hydrogen;

W^(11') and W^(12') are methyl or are taken together with the nitrogenatom to which they are attached to form a morpholino or piperidine ring;

W^(15') and W^(16') are hydrogen, hydroxy, acetoxy, at least one beinghydroxy or acetoxy; and

W^(17') is alkyl of from 1 to 3 carbon atoms.

The methadone derivatives will for the most part have the followingformula: ##STR11## wherein:

one of W^(11") or W^(17") is X**;

X**, A**, and n' have been defined previously;

φ is phenyl;

when other than X**

W^(11") is methyl; and

W^(17") is propyl.

The metabolites of methadone and close analogs will for the most parthave the following formula: ##STR12## wherein:

any one of the W groups can be -X*, X*, A* and n have been definedpreviously;

φ is phenyl;

W^(10"') is hydrogen, hydroxyl, methoxyl or acetoxyl, that is of from 0to 2 carbon atoms, and except when hydrogen of from 1 to 2 oxygen atoms;

W^(11"') is hydrogen, methyl, or a free valence joined with W^(15"') ;

W^(12"') is an unshared pair of electrons;

W^(13"') is hydrogen or methyl;

W^(15"') is hydrogen, hydroxy, or taken together with W^(11"') forms adouble bond between the nitrogen atom and the carbon atom to whichW^(11"') and W^(15"') are respectively attached; and

W^(17"') is alkyl of from one to three carbon atoms, usually two carbonatoms, or may be taken together with W^(15"') to form alkylidenyl offrom 1 to 3 carbon atoms, usually 2 carbon atoms.

Preferred compounds are those where W^(11"') or W^(17"') are X*,particularly W^(17"'), with W^(11"') as methyl.

Illustrative compounds which may be linked to an enzyme includephenylbenzyl(1-dimethylamino-2-propyl)methyl succinate,phenylbenzyl(1-dimethylamino-2-propyl)methyl oxalate,diphenyl(2-dimethylamino-1-propyl)methyl maleate, O-carboxymethyl4,4-diphenyl-7-dimethylamino-2-heptanone oxime,4,4-diphenyl-7-dimethylamino-3-octyl succinate,N-(2,2-diphenyl-3-methyl-4-morpholinobutyryl)glycine,3-ethyl-4,4-diphenyl-6-dimethylaminohept-2-enoic acid,6-keto-7,7-diphenyl-9-diphenyl-9-(dimethylamino) decanoic acid,N-carboxymethyl 2-ethyl-3,3-diphenyl-5-methylpyrrolidine.

Meperidine

The third group of compounds which have narcotic activity and aremeperidine or meperidine analogs, have for the most part the followingformula: r1 ? ##STR13## wherein:

any one of the W groups can be -X*;

X*, A*, and n have been defined previously;

W²⁰ is hydrogen;

W²¹ is hydrogen, alkyl of from 1 to 3 carbon atoms, e.g., methyl,aminophenylakyl, e.g., β-(p-aminophenyl)ethyl, or phenylaminoalkyl,e.g., phenylaminopropyl, (alkyl of from 2 to 3 carbon atoms);

W²² is alkoxy of from 1 to 3 carbon atoms, e.g, ethoxy; and

W²³ is hydrogen or methyl.

Illustrative compounds are meperidine, alphaprodine, alvodine andanileridine.

Preferred compounds are those where W²¹ or W²² is -X* or a hydrogen ofW²¹ is replaced with -X*.

Indole Alkaloids

A second group of ligands of interest are based on tryptamine and comewithin the class of indole alkaloids, more specifically ergot alkaloids.These compounds will have the following minimal structure: ##STR14##wherein the free valences are satisfied by a variety of groups,primarily carbon and hydrogen, although other substituents may bepresent such as carboxyl groups, hydroxyl groups, keto groups, etc. Themost common member of this class which finds use is lysergic acid,primarily as its diethylamide. Other members of the indole alkaloidfamily which can also be assayed for are the strychnine group and theindolopyridocoline group, which finds yohimbine and reserpine asmembers.

The enzyme substituted indole alkaloids will have the following formula:##STR15##wherein m, X and A have been defined previously.

Other groups of alkaloids include the steroid alkaloids, the iminazolylalkaloids, the quinazoline alkaloids, the isoquinoline alkaloids, thequinoline alkaloids, quinine being the most common, and the diterpenealkaloids.

For the most part, the alkaloids bonded to an enzyme will be of fromabout 300 to 1,500 molecular weight, more usually of from about 400 to1,000 molecular weight. They are normally solely composed of carbon,hydrogen, oxygen, and nitrogen; the oxygen is present as oxy and oxo andthe nitrogen present as amino or amido.

Catecholamines

The first group in this category are catecholamines of the formula:##STR16## wherein:

any one of the W groups can be -X*;

X*, A* and n have been defined previously;

W³⁰ is hydrogen or alkyl of from 1 to 3 carbon atoms, e.g., methyl;

W³¹ is hydrogen, or alkyl of from 1 to 3 carbon atoms, e.g., methyl;

W³² and W³³ are hydrogen;

W³⁴ is hydrogen, hydroxy, dimethoxycarboxyphenacyl, anddimethoxy-α-phthalidyl;

W³⁵ and W³⁶ are hydrogen, one of which may be taken with W³¹ to form abond, and when W³¹ and W³⁵ are taken together, each of W³² and W³³, andW³⁰ and W³⁶ may be taken together to form a double bond;

W³⁷ is hydrogen or alkoxy of from 1 to 3 carbon atoms; e.g., methoxy;

W³⁸ and W³⁹ are hydroxy or alkoxy of from 1 to 3 carbon atoms, e.g.,methoxy.

Illustrative compounds include cotainine, narceine, noscapine andpapaverine.

Preferred compounds are where W³⁰, W³⁸ or W³⁹ are -X* or have a hydrogenreplaced with -X*.

A group of compounds related to the catecholamines are epinephrine,amphetamines and related compounds. These compounds have the formula:##STR17## wherein:

any one of the W groups can be -X*;

X*, A* and n have been defined previously;

W⁴⁰ and W⁴¹ are hydrogen or alkyl of from 1 to 3 carbon atoms, e.g.,methyl and isopropyl, preferably one is hydrogen;

W⁴² is hydrogen, alkyl of from 1 to 3 carbon atoms, e.g., methyl andethyl, or may be taken together with W⁴⁰ to form a ring having sixannular members with the nitrogen as the only heteroatom;

W⁴³ is hydrogen, hydroxyl, carbomethoxy, or may be taken together withW⁴⁰ to form a morpholine ring;

W⁴³ is carbomethoxy, when W⁴⁰ and W⁴² are taken together to form apiperidine ring; and

W⁴⁴ and W⁴⁵ are hydrogen, hydroxyl or alkoxyl of from 1 to 3 carbonatoms.

Illustrative compounds which can be bonded to an enzyme are ephedrine,epinephrine, L-dopa, benzidrine (amphetamine), paredrine,methamphetamine, methyl phenidate and norephedrine.

Illustrative compounds which can be linked to an enzyme include3-(3',4'-dihydroxyphenyl)-3-hydroxypropionic acid,N-(β-(β,3,4-trihydroxyphen)ethyl) N-methyl glycine,N-(1-phenyl-2-propyl)oxalamic acid,O-(1-phenyl-2-methylamino-1-propyl)glycolic acid,p-(2-methylaminopropyl-1)phenoxyacetic acid, N-(1'-phenyl-2'-propyl)glycine, 4-methylamino-4-phenylvaleric acid,para-(2-aminopropyl-1)phenoxyacetic acid, 4-methylamino-5-phenylvalericacid, and 3-amino-4-phenylbutyric acid.

Where W⁴⁴ and W⁴⁵ are hydrogen, preferred compounds will have thefollowing formula: ##STR18## wherein:

any one of the W groups can be -X*;

X*, A* and n have been defined previously;

W^(40') and W^(41') are hydrogen or alkyl of from 1 to 3 carbon atoms,preferably one is hydrogen;

W^(42') is hydrogen, methyl or may be taken together with W^(40') toform a piperidine ring;

W^(43') is hydrogen, hydroxyl or carbomethoxy; and

W^(46') is hydrogen.

Where W⁴⁴ and W⁴⁵ are oxy, the preferred compounds have the followingformula: ##STR19## wherein:

any one of the W groups can be -X*;

X*, A* and n have been defined previously;

W^(40"), W^(41"), and W^(42") are hydrogen or methyl;

W^(43") is hydrogen or hydroxyl; and

W^(44") and W^(45") are hydroxyl or methoxy.

Closely related compounds to the amphetamines are those where asaturated five or six membered ring is substituted for the phenyl ring.These compounds will have the following formula: ##STR20## wherein:

any one of the W groups is -X*;

X*, A* and n have been defined previously;

W^(40'-41') have been defined above;

W^(42') is hydrogen or methyl;

W^(43') is hydrogen or hydroxyl;

W^(46') is hydrogen; and

b is an integer of from four to five.

Of particular interest are those amphetamines bonded to enzymes of thefollowing formula: ##STR21## wherein one of W^(40"'), W^(42"'), andW^(44"') is -X**; when other than -X**

W^(40"') is hydrogen;

W^(42"') is methyl; and

W^(44"') is hydrogen;

W^(41"') is hydrogen or methyl;

X** is -Z-CO-, wherein Z is hydrocarbylene of from 1 to 7 carbon atoms,usually aliphatic, having from 0 to 1 site of ethylenic unsaturaction,with the proviso that when W^(44"') is -X**, -X** is -O-Z-CO-;

A** and n' have been defined previously.

Barbiturates

A wide class of synthetic drugs which finds extensive and frequent abuseare the barbiturates. These compounds are synthetically readilyaccessible and their use only difficultly policed. The compounds whichfind use will come within the following formula: ##STR22## wherein:

any one of the W groups can be -X*;

X*, A*, and n have been defined previously;

W⁵⁰ is hydrogen, alkyl of from 1 to 3 carbon atoms, e.g., methyl oralkali metal, e.g., sodium;

W⁵¹ and W⁵² are hydrogen, alkyl, alkenyl, cycloalkyl, cycloalkenyl, oraryl hydrocarbon of from 1 to 8, more usually 1 to 6 carbon atoms, e.g.,ethyl, n-butyl, α-methylbutyl, isoamyl, allyl, Δ¹ -cyclohexenyl, andphenyl;

W⁵³ is hydrogen, or alkali metal, e.g., sodium;

W⁵⁴ is oxygen or sulfur.

Illustrative compounds are veronal, medinal, luminal, prominal, soneryl,nembutal, amytal, dial, phenadorn, seconal, evipan, phenobarbital andpentothal.

Preferred compounds would have W⁵⁰ or W⁵¹ or a hydrogen of W⁵⁰ or W⁵¹ as-X*. Also preferred is when one of W⁵¹ and W⁵² is hydrocarbyl of from 2to 8 carbon atoms.

Illustrative compounds which may be linked to an enzyme include5,5-diethyl-1-carboxymethylbarbituric acid,5-ethyl-5-n-butyl-1-succinoylbarbituric acid,5-ethyl-5-phenyl-1-(N'-(2'''-chloroethyl)-2"-aminoethyl)barbituric acid,5-(2'-carboxy-Δ^(1'2') -cyclohexenyl)-1,5-dimethylbarbituric acid,N-carboxymethyl phenobarbital, 5-(γ-crotonicacid)-5-(2'-pentyl)-barbituric acid, 5-(p-aminophenyl)-5-ethylbarbituricacid, 5-(5'-pentanoic acid)-5-(2'-pentyl)barbituric acid, and1-methyl-5-ethyl-5-(p-carboxyphenyl)barbituric acid.

Of particular interest are those barbiturates bonded to an enzyme of theformula: ##STR23## wherein one of W^(50') and W^(51') is -X**; whenother than -X**:

W^(50') is hydrogen, methyl or alkali metal, e.g., sodio; and

W^(51') is hydrocarbon of from 1 to 8 carbon atoms, having from 0 to 1site of ethylenic unsaturation;

W^(52') is hydrocarbon of from 2 to 8 carbon atoms, having from 0 to 1site of ethylenic unsaturation;

X** is -Z-CO-, wherein Z is hydrocarbylene of from 1 to 7 carbon atoms,usually aliphatic, having from 0 to 1 site of ethylenic unsaturation;

A** and n' have been defined previously.

Glutethimide

Another compound of interest is glutethimide, wherein the enzyme boundanalog will have the following formula: ##STR24## wherein:

any one of the W groups can be -X*;

X*, A* and n have been defined previously;

W^(a50) and W^(a51) are hydrogen; and

W^(a52) is lower alkyl of from 1 to 3 carbon atoms, e.g., ethyl.

Cocaine

A drug of significant importance in its amount of use is cocaine. Theenzyme bound cocaine or cocaine metabolites or analogs, such asecgonine, will for the most part have the following formula: ##STR25##wherein:

any one of the W groups can be -X*;

X*, A* and n have been defined previously;

W⁵⁵ is hydroxy, methoxy, amino or methylamino;

W⁵⁶ is hydrogen or benzoyl; and

W⁵⁷ is hydrogen or alkyl of from 1 to 3 carbon atoms, e.g., methyl.

Of particular interest are those ecgonine derivatives (including cocainederivatives) of the formula: ##STR26## wherein one of W^(56') andW^(57') is -X**; when other than -X**:

W^(56') is hydrogen or benzoyl; and

W^(57') is methyl;

W^(55') is hydroxy or methoxy;

X** is ##STR27##

wherein Z^(a) is methylene or carbonyl; or -Z-CO- wherein Z ishydrocarbylene of from 1 to 7 carbon atoms, usually aliphatic, havingfrom 0 to 1 site of ethylenic unsaturation;

A** and n' have been defined previously.

Diphenyl Hydantoin

Another compound of interest is the antiepileptic drug diphenylhydantoin. This compound and its analogs will have the followingformula: ##STR28## wherein:

any one of the W groups can be -X*;

X*, A* and n have been defined previously;

φ is phenyl;

W^(a60), W^(a61) and W^(a62) are hydrogen.

Marijuana

Because of its ready availability and widespread use,tetrahydrocannabinol (the active ingredient of marijuana) and itscongeners, cannabidiol and cannabinol and their metabolites arecompounds of great interest, where a simple assay method would be ofimportance. The compounds which find use as analogs have the followingformula: ##STR29## wherein:

any one of the W groups can be -X*;

X*, A* and n have been defined previously;

W^(a10) is hydrogen or carboxyl;

W^(a11) is hydroxyl or methoxyl;

W^(a12) is hydrogen;

W^(a13) is pentyl or hydroxypentyl;

W^(a14) is hydrogen, methyl, or the two W^(a14) 's may be taken togetherto form a carbocyclic ring of from 5 to 6 annular members; and

W^(a15) is methyl, hydroxymethyl or carboxyl.

Tranquilizers

A number of compounds have tranquilizer effects and because of theirmisuse or abuse do provide opportunities where the determination couldbe of use.

The first tranquilizer of interest is Meprobamate, also known as Miltownor Equanil. This compound and related analogs have the followingformula: ##STR30## wherein:

any one of the W groups can be -X*;

X*, A* and n have been defined previously;

W^(a25) and W^(a26) are amino.

The next group of tranquilizers are benzdiazocycloheptanes and are knownas Librium, Valium, Diazepam, or Oxazepam. These compounds and theirrelated analogs will have the following formula: ##STR31## wherein:

any one of the W groups can be -X*;

X*, A*, and n have been defined previously;

W^(a30) and W^(a35) are hydrogen;

W^(a31) is hydrogen, lower alkyl of from 1 to 3 carbon atoms, e.g.,methyl, or may be taken together with W^(a32) to form a double bondbetween the carbon and the nitrogen;

W^(a33) is amino or lower alkylamino of from 1 to 3 carbon atoms, e.g.,methylamino, or may be taken together with W^(a32) to form a carbonyl;

W^(a34) is hydrogen or hydroxyl; and

W^(a36) is oxy or an unshared pair of electrons.

The next group of compounds are the phenothiazines of whichchlorpromazine is a member. These compounds will for the most part havethe following formula: ##STR32## wherein:

any one of the W groups can be -X*;

X*, A*, and n have been defined previously;

W^(a40) is hydrogen, alkyl of from 1 to 6 carbon atoms,dialkylaminoalkyl of from 4 to 8 carbon atoms, e.g.,3-(dimethylamino)propyl; N-hydroxyalkyl (alkyl of from 2 to 3 carbonatoms), N'-piperazinoalkyl (alkyl of from 2 to 3 carbon atoms), e.g.,N-hydroxyethyl N'-piperazinopropyl; N-alkyl (alkyl of from 1 to 3 carbonatoms) N'-piperazinoalkyl (alkyl of from 2 to 3 carbon atoms), e.g.,N-methyl N'-piperazinopropyl; and 2-(N-alkyl)-piperidinoalkyl, whereinthe N-alkyl is of from 1 to 3 carbon atoms and the other alkyl is offrom 2 to 3 carbon atoms, e.g., 2-(N-methyl)-piperidinoethyl, therebeing at least two carbon atoms between the heteroatoms;

W^(a41) is hydrogen, chloro, trifluoromethyl, alkylmercapto of from 1 to3 carbon atoms, e.g., methylmercapto and acyl of from 1 to 3 carbonatoms, e.g., acetyl; and

W^(a42) and W^(a43) are hydrogen.

Amino Acids, Polypeptides and Proteins

The next group of compounds are the amino acids, polypeptides andproteins. For the most part, the amino acids range in carbon contentfrom 2 to 15 carbon atoms, and include a variety of functional groupssuch as mercapto, dithio, hydroxyl, amino, guanidyl, pyrrolidinyl,indolyl, imidazolyl, methylthio, iodo, diphenylether, hydroxyphenyl,etc. These, of course, are primarily the amino acids related to humans,there being other amino acids found in plants and animals.

Polypeptides usually encompass from about 2 to 100 amino acid units(usually less than about 12,000 molecular weight). Larger polypeptidesare arbitrarily called proteins. Proteins are usually composed of from 1to 20 polypeptide chains, called subunits, which are associated bycovalent or non-covalent bonds. Subunits are normally of from about 100to 400 amino acid groups (˜10,000 to 50,000 molecular weight).

Individual polypeptides and protein subunits will normally have fromabout 2 to 400, more usually from about 2 to 300 recurring amino acidgroups. Usually, the polypeptides and protein subunits of interest willbe not more than about 50,000 molecular weight and greater than about750 molecular weight. Any of the amino acids may be used in preparingthe polypeptide. Because of the wide variety of functional groups whichare present in the amino acids and frequently present in the variousnaturally occurring polypeptides, the enzyme bonded compound can bebonded to any convenient functionality. Usually, the enzyme bondedcompound can be bonded to a cysteine, lysine or arginine, tyrosine orhistidine group, although serine, threonine, or any other amino acidwith a convenient functionality, e.g., carboxy and hydroxy, may be used.

For the most part, the enzyme-labeled polypeptides will have thefollowing formula: ##STR33## wherein X and A have been definedpreviously, and R is an amino acid residue, r being an integer of from 1to 1,000, more usually of from 1 to 500, and most commonly of from 2 to100. r' is an integer of at least one and not greater than the molecularweight of the polypeptide divided by 2,000.

Illustrative amino acids include glycine, alanine, serine, histidine,methionine, hydroxyproline, tryptophan, tyrosine, thyroxine, ornithine,phenylalanine, arginine, and lysine. Polypeptides of interest are ACTH,oxytocin, lutenizing hromone, insulin, Bence-Jones protein, chlorionicgonadotropin, pituitary gonadotropin, growth hormone, rennin, thyroxinebonding globulin, bradykinin, angiotensin, follicle stimulating hormone,etc.

In certain instances, it will be desirable to digest a protein and assayfor the small polypeptide fragments. The concentration of the fragmentmay then be related to the amount of the original protein.

Steroids

Another important group of compounds which find use in this inventionare the steroids, which have a wide range of functionalities dependingon their function in the body. In addition to the steroids, are thesteroidmimetic substances, which while not having the basic polycyclicstructure of the steroid, do provide some of the same physiologicaleffects.

The steroids have been extensively studied and derivatives preparedwhich have been bonded to antigenic proteins for the preparation ofantibodies to the steroids. Illustrative compounds include:17β-estradiol-6-(O-carboxymethyl-oxime)-BSA (bovine serum albumin)(Exley, et al, Steroids 18 593, (1971); testosterone-3-oxime derivativeof BSA (Midgley, et al, Acta Endocr. 64 supplement 147, 320 (1970)); andprogesterone-3-oxime derivative of BSA (Midgley, et al, ibid.)

For the most part, the steroids used have the following formula:##STR34## wherein m, X and A have been defined previously. Usually, theenzyme will be bonded to the A, B, or C rings, at the 2, 3, 4, 6 or 11positions, or at the 16 or 17 positions of the D ring or on the sidechains at the 17 position. Of particular interest is where X is bondedto the 6 position. The rings may have various substituents; particularlymethyl groups, hydroxyl groups, oxocarbonyl groups, ether groups, andamino groups. Any of these groups may be used to bond the enzyme to thebasic ring structure. For the most part, the steroids of interest willhave at least one, usually 1 to 6, more usually 1 to 4 oxygenfunctionalities, e.g., alcohol, ether, esters, or keto. In addition,halo substituents may be present. The steroids will usually have from 18to 27 carbon atoms, or as a glycoside up to 50 carbon atoms.

The rings may have one or more sites of unsaturation, either ethylenicor aromatic and may be substituted at positions such as the 6, 7 and 11positions with oxygen substituents. In addition, there may be methylgroups at the 10 and 13 positions. The position marked with a Z, 17, maybe and will be varied widely depending on the particular steroid. Zrepresents two monovalent groups or one divalent group and may be acarbonyl oxygen, an hydroxy group, an aliphatic group of from 1 to 8carbon atoms, including an acetyl group, an hydroxyacetyl group, carboxyand carboxyalkyl of from 2 to 6 carbon atoms, an acetylenic group offrom 2 to 6 carbon atoms or halo substituted alkyl or oxygenated alkylgroup or a group having more than one functionality, usually from 1 to 3functionalities.

For the second valence of Z, there may be a H or a second group,particularly hydroxyl, alkyl, e.g., methyl, hydrodyalkyl, e.g.,hydroxymethyl; halo, e.g., fluoro or chloro, oxyether; and the like.

These steroids find use as hormones, male and female (sex) hormones,which may be divided into oestrogens, gestogens, antrogens,adrenocortical hormones (glucocorticoids), bile acids, cardiotonicglycosides and aglycones, as well as saponins sapogenins.

Steroid mimetic sutstances, particularly sex hormones are illustrated bydiethyl stilbestrol.

The sex hormones of interest may be divided into two groups; the malehormones (androgens) and the female hormones (oestrogens).

The androgens which find use will have the following formula: ##STR35##wherein:

any one of the W groups can be -X*;

X*, A* and n have been defined previously;

W⁶⁰ is hydrogen, or hydroxyl;

W⁶¹ is hydrogen, methyl or hydroxyl (when two groups bonded to the samecarbon atom are hydroxyl, oxo is intended);

W⁶² and W⁶³ are hydrogen or hydroxyl, at least one of W⁶⁰⁻⁶³ is hydroxy(either as hydroxy or oxo);

W⁶⁴ is hydrogen, or two W^(64') s may be taken together to form a doublebond;

W⁶⁵ is methyl; and

W⁶⁶ is hydrogen.

Illustrative compounds which may be bonded to an enzyme includetestosterone, androsterone, isoandrosterone, etiocholanolone,methyltestosterone and dehydroisoandrosterone.

Illustrative compounds which may be linked to an enzyme includeN-carboxymethoxy testosteroneimine, 17-monotestosteronyl carbonate,androsteronyl succinate, testosteronyl maleate, O³ -carboxymethyl O¹⁷-methyl androst-5-ene-3β, 17β-diol, testosterone O-carboxypropyl oximeand androsteronyl carbonate.

The oestrogens have an aromatic A ring and for the most part have thefollowing formula: ##STR36## wherein:

any one of the W groups can be -X*;

X*, A* and n have been defined previously;

W⁷⁰ and W⁷¹ are hydrogen, ethinyl or hydroxyl (when two hydroxyls arebonded to the same carbon atom, oxo is intended);

W⁷² is hydrogen or hydroxyl;

W⁷³ is hydroxyl or alkoxyl of from 1 to 3 carbon atoms;

W⁷⁴ is hydrogen or two W^(74') s may be taken together to form a doublebond; and

W⁷⁵ is hydrogen.

Illustrative compounds which may be bonded to an enzyme are equilenin,β-estradiol, estrone, estriol, and 17-α-ethinyl-estradiol.

Illustrative compounds which may be linked to an enzyme include3-carboxymethyl estradiol, b 2-chloromethylestrone, estrone glutarate,O-carboxymethyloxime of 6-ketoestradiol, equilenyl N-carboxymethylthiocarbamate.

Another class of hormones are the gestogens which have the followingformula: ##STR37## wherein:

any one of the W groups can be -X*;

X*, A* and n have been defined previously;

W⁸⁰ and W⁸¹ are hydrogen or hydroxyl, at least one being hydroxyl (wheretwo hydroxyl groups are bonded to the same carbon atom, oxo isintended);

W⁸² is hydrogen or hydroxyl;

W⁸³ and W⁸⁴ are hydrogen or hydroxyl, at least one being hydroxyl; and

W⁸⁵ is hydrogen, or two W⁸⁵ 's may be taken together to form a doublebond.

Illustrative compounds which may be bonded to an enzyme includeprogesterone, pregnenolone, allopregnane-3a:20a-diol andallopregnan-3a-ol-20-one.

Illustrative compounds which may be linked to an enzyme include20-progesterone O-carboxymethyl oxime,pregn-4-en-20-on-3-ylidinylmethylenecarboxylic acid, O-carboxymethylprogesterone 3-oxime, pregnenolonyl tartrate, O-pregnenolonyl tartrate,O-pregnenolonyl lactic acid, and allopreganane-3-carboxymethyl-20-ol.

The next important group of steroids is the corticosteroids whichincludes both the mineralcorticoids and the glucocorticoids. Thesecompounds have the following formula: ##STR38## wherein:

any one of the W groups can be -X*;

X*, A* and n have been defined previously;

W⁹⁰ is hydrogen or hydroxyl;

W⁹¹ and W⁹² are hydrogen or hydroxyl, at least one of which is hydroxyl(when two hydroxyl groups are bonded to the same carbon atom, oxo isintended);

W⁹³ is hydrogen or hydroxyl;

W⁹⁴, W⁹⁵, W⁹⁶, and W⁹⁷ are hydrogen or hydroxyl, at least one of W⁹⁴ andW⁹⁵ is hydroxyl;

W⁹⁸ is methyl or formyl; and

W⁹⁹ is hydrogen or two W⁹⁹ 's may be taken together to form a doublebond.

Illustrative compounds which may be bonded to an enzyme are17-hydroxydioxycorticosterone (Compound S), deoxycorticosterone,cortisone, corticosterone, 11-dihydrocortisone (Compound F), cortisol,prednisolone and aldosterone.

Illustrative compounds which may be linked to an enzyme include O²¹-carboxymethyl corticosterone, N-carboxymethyl 21-carbamate cortisol,21-cortisone succinate, 21-deoxocorticosterone succinate, and O¹⁷-methyl, O²¹ -carboxymethyl cortisone.

An additional steroid family is the cardiotonic glycosides and aglyconesof which digitalis is an important member. The basic compound isdigitoxigenin, which is also found as the glycoside. The compounds ofinterest have the following formula: ##STR39## wherein:

any one of the W groups can be -X*;

X*, A* and n have been defined previously;

W^(a1), W^(a2), W^(a3) and W^(a4) are hydrogen, hydroxyl, or aglycoside, at least one being hydroxyl or a sugar, mostly as aglycoside. The sugars include xylose, glucose, cymarose, rhamnose, andgalactose.

Also of interest are the saponins and sapogenins derived from plants.These compounds have a spiro ring structure at C₂₂.

Vitamins

The next group of compounds are the vitamins. Chemically, the vitaminsdo not provide a single chemical class, varying greatly in structure,but being classified as a group as to function. The vitamins include,vitamin A, which is a carotene, the B vitamin group which includesriboflavin, thiamine, niacin, pyridoxine, pantothenic acid, biotin,folic acid, and cyanocobalamine (Vitamin B₁₂); ascorbic acid (VitaminC); the D vitamins which are steroidal derived; tocopherol (Vitamin E);and phytyl-1,4-naphthoquinone (Vitamin K).

Sugars

The next group of compounds are the sugars and saccharides. Thesaccharides are combinations of various sugars to form dimers, trimersand high molecular weight polymers, referred to as polysaccharides.

Prostaglandin

Another group of compounds of biological importance are theprostaglandins. These compounds when bonded to enzymes have for the mostpart the following formula: ##STR40## wherein:

any one of the W groups can be -X*;

X*, A* and n have been defined previously; W^(a20) is hydrogen orhydroxyl;

W^(a21) and W^(a22) are hydrogen or hydroxyl, (where two hydroxyl groupsare bonded to the same carbon atom, oxo is intended);

W^(a23) is hydrogen or hydroxyl; and

W^(a24) is hydroxyl, amino or an oxy group of from 1 to 6 carbon atoms,e.g., alkoxy.

Miscellaneous

Included in this group are the antibiotics such as penicillin,chloromycetin, actinomycetin, tetracycline, terramycin, and nucleicacids or derivatives, such as nucleosides and nucleotides.

Also of interest is serotonin which is3-(2'-aminoethyl)-5-hydroxyindole. -X* may be bonded at either of theamino nitrogen atoms or the hydroxyl group.

Of course, many of the compounds which are of interest undergo metabolicchanges, when introduced into a vertebrate. The particular physiologicalfluid which is tested may have little, if any of the original compound.Therefore, the original presence of the compound might only bedetectable as a metabolite. In many instances, the metabolite may be theglucuronide, either oxy or oxo derivative of the original compound. Inother instances, the original compound may have undergone oxidation,e.g., hydroxylation, reduction, acetylation, deamination, amination,methylation or extensive degradation. Where the metabolite still retainsa substantial portion of the spatial and polar geometry of the originalcompound, it will be frequently possible to make the ligand analog basedon either the original compound or metabolite. Where the metabolite isdistinctively different than the original compound, the ligand analogwill be based on the metabolite.

Of particular interest as metabolites, particularly of the steroids, arethe sulfates and glucuronides.

Besides metabolites of the various drugs, hormones and other compoundspreviously described, of significant interest are metabolites whichrelate to diseased states. Illustrative of such compounds are spermine,galactose, phenylpyruvic acid and porphyrin Type 1, which are believedto be diagnostic of certain tumors, galactosemia, phenylketonuria andcongenital porphyra, respectively.

Two compounds of interest which are metabolites of epinephrine arevanillylmandelic acid and homovanillic acid. With these compounds,either the hydroxyl or carboxyl groups can be used as the site for -X*.

Another general category of interest is the pesticides, e.g.,insecticides, fungicides, bacteriocides and nematocides. Illustrativecompounds include phosphates such as malathion, DDVP, dibrom;carbamates, such as Sevin, etc.

Since many of the biologically active materials are active in only onestereoisomeric form, it is understood that the active form is intendedor the racemate, where the racemate is satisfactory and readilyavailable. The antibodies will be specific for whatever form is used asthe hapten.

Enzymes (A)

Enzymes vary widely in their substrates, cofactors, specificity,ubiquitousness, stability to temperature, pH optimum, turnover rate, andthe like. Other than inherent factors, there are also the practicalconsiderations, that some enzymes have been characterized extensively,have accurate reproducible assays developed, and are commerciallyavailable. In addition, for the purposes of this invention, the enzymesshould either be capable of specific labelling or allow for efficientsubstitution, so as to be useful in the subject assays. By specificlabelling is intended selective labelling at a site in relationship tothe active site of the enzyme, so that upon binding of the receptor tothe ligand, the enzyme is satisfactorily inhibited. By allowing forefficient substitution to be useful in the subject assay, it is intendedthat the enzyme be inhibited sufficiently when the ligand is bound tothe receptor, and that the degree of substitution required to achievethis result does not unreasonably diminish the turnover rate for theenzyme nor substantially change the enzyme's solubility characteristics.

For the standpoint of operability, a very wide variety of enzymes can beused. But, as a practical matter, there will be a number of groups ofenzymes which are preferred. Employing the International Union ofBiochemists (I.U.B.) classification, the oxidoreductases (1.) and thehydrolases (3.) will be of greatest interest, while the lyases (4.) willbe of lesser interest. Of the oxidoreductases, the ones acting on theCHOH group, the aldehyde or keto group, or the CH-NH₂ group as donors(1.1, 1.2, and 1.4 respectively) and those acting on hydrogen peroxideas acceptor (1.11) will be preferred. Also, among the oxidoreductases aspreferable will be those which employ nicotinamide adenine dinucleotide,or its phosphate or cytochrome as an acceptor, namely 1.x.1 and 1.x.2,respectively under the I.U.B. classification. Of the hydrolases, ofparticular interest are those acting on glycosyl compounds, particularlyglycoside hydrolases, and those acting on ester bonds, both organic andinorganic esters, namely the 3.1 and 3.2 groups respectively, under theI.U.B. classification. Other groups of enzymes which might find use arethe transferases, the lyases, the isomerases, and the ligases.

In choosing an enzyme for commercialization, as compared to a single orlimited use for scientific investigation, there will be a number ofdesireable criteria. These criteria will be considered below.

The enzyme should be stable when stored for a period of at least threemonths, and preferably at least six months at temperatures which areconvenient to store in the laboratory, normally -20° C or above.

The enzyme should have a satisfactory turnover rate at or near the pHoptimum for binding to the antibody, this is normally at about pH 6 -10, usually 6.0 to 8.0. Preferably, the enzyme will have the pH optimumfor the turnover rate at or near the pH optimum for binding of theantibody to the ligand.

A product should be either formed or destroyed as a result of the enzymereaction which absorbs light in the ultraviolet region or the visibleregion, that is in the range of about 250-750 nm, preferably 300-600 nm.

Preferably, the enzyme should have a substrate (including cofactors)which has a molecular weight in excess of 300, preferably in excess of500, there being no upper limit. The substrate may either be the naturalsubstrate, or a synthetically available substrate.

Preferably, the enzyme which is employed or other enzymes, with likeactivity, will not be present in the fluid to be measured, or can beeasily removed or deactivated prior to the addition of the assayreagents. Also, one would want that there not be naturally occurringinhibitors for the enzyme present in fluids to be assayed.

Also, although enzymes of up to 600,000 molecular weight can beemployed, usually relatively low molecular weight enzymes will beemployed of from 10,000 to 300,000 molecular weight, more usually fromabout 10,000 to 150,000 molecular weight, and frequently from 10,000 to100,000 molecular weight. Where an enzyme has a plurality of subunitsthe molecular weight limitations refer to the enzyme and not to thesubunits.

For synthetic convenience, it is preferable that there be a reasonablenumber of groups to which the ligand may be bonded, particularly aminogroups. However, other groups to which the ligand may be bonded includehydroxyl groups, thiols, and activated aromatic rings, e.g., phenolic.

Therefore, enzymes will preferably be chosen which are sufficientlycharacterized so as to assure the availability of sites for linking,either in positions which allow for inhibition of the enzyme when theligand is bound to antibody, or there exist a sufficient number ofpositions as to make this occurrence likely.

A list of common enzymes may be found in Hawk, et al, PracticalPhysiological Chemistry, McGraw-Hill Book Company, New York (1954),pages 306 to 307. That list is produced in total as follows, includingthe source of the enzyme, the substrate and the end products.

    __________________________________________________________________________    Name & Class  Distribution                                                                            Substrate                                                                              End-products                                 __________________________________________________________________________    Hydrolases                                                                    Carbohydrases           Carbohy-                                                                      drates                                                1. Amylase    Pancreas, sal-                                                                          Starch, dex-                                                                           Maltose and                                                iva, malt, etc.                                                                         trin, etc.                                                                             dextrins                                     2. Lactase    Intestinal juice                                                                        Lactose  Glucose and                                                and mucosa         galactose                                    3. Maltase    Intestinal juice,                                                                       Maltose  Glucose                                                    yeast, etc.                                                     4. Sucrase    Intestinal juice   Glucose and                                                yeast, etc.                                                                             Sucrose  fructose                                     5. Emulsion   Plants    β-Gluco-                                                                          Glucose, etc.                                                        sides                                                 Nucleases               Nucleic acid                                                                  and deriva-                                                                   tives                                                 1. Polynucleo-                                                                              Pancreatic juice                                                                        Nucleic  Nucleotides                                    tidase      intestinal juice                                                                        acid                                                                etc.                                                            2. Nucleoti-  Intestinal juice                                                                        Nucleotides                                                                            Nucleotides and                                dase        and other tissues  phosphoric acid                              3. Nucleotidase                                                                             Animal tissues                                                                          Nucleotides                                                                            Carbohydrate                                                                  and bases                                    Amidases                Amino com-                                                                    pounds and                                                                    amides                                                1. Arginase   Liver     Arginine Ornithine and                                                                 urea                                         2. Urease     Bacteria, soy-                                                                          Urea     Carbon dioxide                                             bean, jack bean    and ammonia                                                etc.                                                            3. Glutami-   Liver, etc.                                                                             Glutamine                                                                              Glutamic acid                                nase                             and ammonia                                  4. Transaminase                                                                             Animal tissues                                                                          Glutamic acid                                                                          α-Ketoglutaric                                                 and oxalacetic                                                                         acid, aspartic                                                       acid, etc.                                                                             acid, etc.                                   Purine Deaminases       Purine basesa                                                                 and deriva-                                                                   tives                                                 1. Adenase    Animal tissues                                                                          Adenine  Hypoxanthine                                                                  and ammonia                                  2. Guanase    Animal tissues                                                                          Guanine  Xanthine and                                                                  ammonia                                      Peptidases              Peptides                                              1. Aminopolypep-                                                                            Yeast, intestines                                                                       Polypeptides                                                                           Simpler pep-                                   tidase      etc.               tides and a-                                                                  mino acids                                   2. Carboxypep-                                                                              Pancreas  Polypeptides                                                                           Simpler pep-                                   tidase                         tides and                                                                     amino acids                                  3. Dipeptidase                                                                              Plant and animal                                                                        Dipeptides                                                                             Amino acids                                                tissues and bac-                                                              teria                                                           4. Prolinase  Animal tissues                                                                          Proline  Proline and                                                and yeast peptides simpler pep-                                                                  tides                                        Proteinases             Proteins                                              1. Pepsin     Gastric juice                                                                           Proteins Proteoses,                                                                    peptones, etc.                               2. Trypsin    Pancreatic juice                                                                        Proteins,                                                                              Polypeptides                                                         proteoses,                                                                             and amino acid                                                       and peptones                                          3. Cathepsin  Animal tissues                                                                          Proteins Proteoses,                                                                    and peptones                                 4. Rennin     Calf stomach                                                                            Casein   Paracasein                                   5. Chymotrypsin                                                                             Pancreatic juice                                                                        Proteins,                                                                              Polypeptides                                                         proteoses                                                                              and amino acid                                                       and peptones                                          6. Papain     Papaya, other                                                                           Proteins,                                                           plants    proteoses,                                                                    and peptones                                          7. Ficin      Fig sap   Proteins Proteoses,                                                                    etc.                                         Esterases               Esters   Alcohols and                                                                  acids                                        1. Lipase     Pancreas, castor                                                                        Fats     Glycerol and                                               bean, etc.         fatty acids                                  2. Esterases  Liver, etc.                                                                             Ethyl buty-                                                                            Alcohols and                                                         rate, etc.                                                                             acids                                        3. Phosphatases                                                                             Plant and animal                                                                        Esters of                                                                              Phosphate and                                              tissues   phosphoric                                                                             alcohol                                                              acid                                                  4. Sulfatases Animal and plant                                                                        Esters of                                                                              Sulfuric acid                                              tissues   sulfuric and alcohol                                                          acid                                                  5. Cholines-  Blood, tissues                                                                          Acetylcho-                                                                             Choline and                                    terase                line     acetic acid                                  Iron Enzymes                                                                  1. Catalase   All living or-                                                                          Hydrogen Water and                                                  ganisms except a                                                                        peroxide oxygen                                                     few species of -                                                                        microorganisms                                        2. Cytochrome All living or-                                                                          Reduced cy-                                                                            Oxidized cyto                                  oxidase     ganisms except a                                                                        tochrome C in                                                                          chrome C and                                               few species of                                                                          the presence                                                                           water                                                      microorganisms                                                                          of oxygen                                             3. Peroxidase Nearly all plant                                                                        A large num-                                                                           Oxidation pro                                              cells     ber of phenols                                                                         duct of                                                              aromatic a-                                                                            substrate                                                            mines, etc.                                                                            and water                                                            in the pre-                                                                   sence of H.sub.2 O.sub.2                              Copper Enzymes                                                                1. Tyrosinase Plant and animal                                                                        Various phe-                                                                           Oxidation pro-                                 (poly-phenol-                                                                             tissues   nolic com-                                                                             duct of sub-                                   oxidase, mono-        pounds   strate                                         phenoloxidase)                                                              2. Ascorbic acid        Ascorbic Dehydroascor-                                  oxidase     Plant tissues                                                                           acid in the                                                                            bic acid                                                             presence of                                                                   oxygen                                                Enzymes Containing                                                            Coenzymes I and/or II                                                         1. Alcohol dehy-                                                                            Animal and plant                                                                        Ethyl alco-                                                                            Acetaldehyde                                   drogenase   tissues   hol and  and other al-                                                        other alco-                                                                            dehydes                                                              hols                                                  2. Malic dehy-                                                                              Animal and plant                                                                        L( ) Malic                                                                             Oxalacetic                                     drogenase   tissues   acid     acid                                         3. Isocritric de-                                                                           Animal and plant                                                                        L-Isocitric                                                                            Oxalosuccinic                                  hydrogenase tissues   acid     acid                                         4. Lactic dehy-                                                                 drogenase   Animal tissues                                                                          Lactic acid                                                                            Pyruvic acid                                               and yeast                                                       Hydroxy-.     Liver, kidneys,                                                                         L-β-Hydroxy-                                                                      Acetoacetic                                    butyric dehydro-                                                                          and heart butyric  acid                                           genase                acid                                                  6. Glucose dehy-                                                                            Animal tissues                                                                          D-Glucose                                                                              D-Gluconic                                     drogenase                      acid                                         7. Robison ester                                                                            Erythrocytes                                                                            Robison es-                                                                            Phosphohexonic                                 dehydrogenase                                                                             and yeast ter (hexo-                                                                             acid                                                                 se-6-phos-                                                                    phate                                                 8. Glycerophos-                                                                             Animal tissues                                                                          Glycero- Phosphogylceri-                                phate dehy-           phosphate                                                                              acid                                           drogenase                                                                   9. Aldehyde de-                                                                 hydrogenase Liver     Aldehydes                                                                              Acids                                        Enzymes which                                                                 Reduce Cytochrome                                                             1. Succinic de-                                                                             Plants, animals                                                                         Succinic Fumaric acid                                   hydrogenase and microor-                                                                            acid                                                    (as ordinarily                                                                            ganisms                                                           prepared)                                                                   Yellow Enzymes                                                                1. Warburg's old                                                                            Yeast     Reduced co-                                                                            Oxidized co-                                   yellow enzyme         enzyme II                                                                              enzyme II and                                                                 reduced yellow                                                                enzyme                                       2. Diaphorase Bacteria, Reduced co-                                                                            Oxidized co-                                               yeasts, higher                                                                          enzyme I enzyme I and                                               plants, and ani-   reduced yel-                                               mals               low diaphorase                               3. Haas enzyme                                                                              Yeast     Reduced co-                                                                            Oxidized co-                                                         enzyme II                                                                              enzyme II and                                                                 reduced yel-                                                                  low enzyme                                   4. Xanthine   Animal tissues                                                                          Hypoxanthine                                                                           Xanthine, uric                                 oxidase               xanthine, al-                                                                          acid, acids,                                                         dehydes, re-                                                                           oxidized co-                                                         duced coen-                                                                            enzyme I, etc.                                                       zyme I, etc.                                                                           In presence                                                                   of air, H.sub.2 O.sub.2                      5. D-amino acid                                                                             Animal tissues                                                                          D-Amino Acids                                                                          α-Keto-acids                             oxidase               + O.sub.2                                                                              + NH.sub.3 + H.sub.2 O.sub.2                 6. L-Amino acid                                                                             Animals, snake                                                                          L-amino acids                                                                          Keto acids                                     oxidases    venoms             and ammonia                                  7. TPN-Cytochrome                                                                           Yeast, liver                                                                            Reduced co-                                                                            Oxidized co-                                   C reductase           enzyme II                                                                              enzyme I and                                                         and cyto-                                                                              reduced cyto-                                                        chrome C chrome C                                     8. DPN Cytochrome                                                                           Liver, yeast                                                                            Reduced co-                                                                            Oxidized co-                                   C reductase           enzyme I and                                                                           enzyme I and                                                         cytochrome C                                                                           reduced cyto-                                                                 chrome C                                     Hydrases                                                                      1. Fumarase   Living organisms                                                                        Fumaric  L-Malic acid                                               in general                                                                              acid + H.sub.2 O                                      2. Aconitase  Animals and                                                                             Citric acid                                                                            cis-Aconitic                                               plants             acid and L-                                                                   isocitric                                                                     acid                                         3. Enolase    Animal tissues                                                                          2-Phospho-                                                                             Phospyruvic                                                and yeast glyceric acid                                                                          acid + H.sub.2 O                             Mutases                                                                       1. Glyoxalase Living organisms                                                                        Methyl gly-                                                                            D (-) Lactic                                               in general                                                                              oxal and acid                                                                 other sub-                                                                    stituted                                                                      glyoxals                                              Desmolases                                                                    1. Zymohexase All cells Fructose-                                                                              Dihydroxy-                                     (aldolase)            1,6-diph-                                                                              acetone ph-                                                          osphate  osphoric acid                                                                 and phospho-                                                                  glyceric acid                                2. Carboxylase                                                                              Plant tissues                                                                           Pyruvic  Acetaldehyde                                                         acid     and CO.sub.2                                 Keto carboxy- Animals, bac-                                                                           β-Keto                                                                            α-Keto acids                             lases       teria, plants                                                                           acids                                                 4. Amino acid de-                                                                           Plants, animals,                                                                        L-Amino  Amines and                                     carboxylases                                                                              bacteria  acids    CO.sub.2                                     5. Carbonic anhy-                                                                           Erythrocytes                                                                            Carbonic CO.sub.2 + H.sub.2 O                           drase                 acid                                                  Other Enzymes                                                                 1. Phosphorylase                                                                            Animal and plant                                                                        Starch or                                                                              Glucose-1-                                                 tissues   glycogen phosphate                                                            and phos-                                                                     phate                                                 2. Phosphohexo-                                                                             Animal and plant                                                                        Glucose-6-                                                                             Fructose-6-                                    isomerase   tissues   phosphate                                                                              phosphate                                    3. Hexokinase Yeast, animal                                                                           Adenosine-                                                                             Adenosined-                                                tissues   triphos- iphosphate                                                           phate    + glucose-                                                                    6-phosphate                                  4. Phosphoglu-                                                                              Plant and animals                                                                       Glucose-1-                                                                             Glucose-6-                                     comutase              phosphate                                                                              phosphate                                    __________________________________________________________________________

Of the various enzymes, the following table indicates enzymes ofparticular interest set forth in accordance with the I.U.B.classification.

1. Oxidoreductases

1.1 Acting on the CH-OH group of donors

1.1.1 With NAD or NADP as acceptor

1. alcohol dehydrogenase

6. glycerol dehydrogenase

26. glyoxylate reductase

27. L-lactate dehydrogenase

37. malate dehydrogenase

49. glucose 6-phosphate dehydrogenase

17. mannitol 1-phosphate dehydrogenase

1.1.2 With cytochrome as an acceptor

3. L-lactate dehydrogenase

1.1.3 With O₂ as acceptor

4. glycose oxidase

9. galactose oxidase

1.2 Acting on the CH-NH₂ group of donors

1.4.3 With O₂ as acceptor

2. L-amino acid oxidase

3. D-amino acid oxidase

1.6 Acting on reduced NAD or NADP as donor

1.6.99 With other acceptors

diaphorase

1.10 Acting on diphenols and related substances as donors

1.10.3 With O₂ as acceptor

1. polyphenol oxidase

3. ascorbate oxidase

1.11 Acting on H₂ O₂ as acceptor

1.11.1

6. catalase

7. peroxidase

3. Hydrolases

3.1 Acting on ester bonds

3.1.1 Carboxylic ester hydrolases

7. cholinesterase

3.1.3 Phosphoric monoester hydrolases

1. alkaline phosphatase

3.1.4 Phosphoric diester hydrolases

3. phospholipase C

3.2 Acting on glycosyl compounds

3.2.1 Glycoside hydrolases

1. α-amylase

4. cellulase

17. lysozyme

23. β-galactosidase

27. amyloglucosidase

31. β-glucuronidase

3.4 Acting on peptide bonds

3.4.2 Peptidyl-amino acid hydrolase

1. carboxypeptidase A

3.4.4 peptidyl-peptide hydrolase

5. α-chymotrypsin

10. papain

3.5 Acting on C-N bonds other than peptide bonds

3.5.1 In linear amides

5. urease

3.6 Acting on acid anhydride bonds

3.6.1 In phosphoryl-containing anhydrides

1. inorganic pyrophosphatase

4. Lyases

4.1 Carbon-carbon lyases

4.1.2 Aldehyde lyases

7. aldolase

4.2 Carbon-oxygen lyases

4.2.1 Hydrolases

1. carbonic anhydrase

4.3 Carbon-nitrogen lyases

4.3.1 Ammonia lyases

3. histidase

Linking Group (X)

The ligand or ligand analog is normally bonded either directly to theenzyme, by a single or double bond, or preferably to a linking group.For those ligands, which are haptens, and for which the receptors areantibodies the ligand will have been bound to a protein for the purposeof preparing the antibodies. Since the antibodies will recognize thatportion of the ligand molecule which extends from the protein,ordinarily the same linking group will be attached on the same site onthe ligand, as was used in bonding the ligand to the protein to providethe antigenic substance.

The functional groups which will be present in the enzyme for linkingare amino (including guanidino), hydroxy, carboxy, and mercapto. Inaddition, activated aromatic groups or imidazole may also serve as asite for linking.

Amino acids having amimo groups available for linking include lysine,arginine, and histidine. Amino acids with free hydroxyl groups includeserine, hydroxyproline, tyrosine and threonine. Amino acids which havefree carboxyl groups include aspartic acid and glutamic acid. An aminoacid which has an available mercapto group is cysteine. Finally, theamino acids which have activated aromatic rings are tyrosine andtryptophan.

In most instances, the preferred linking group will be the amino group.However, there will be situations with certain enzymes, where one of theother linking groups will be preferred.

The ligand, of course, will have a great diversity of functionalitieswhich may be present. In addition, as already indicated, thefunctionalities which are present may be modified so as to form adifferent functionality, e.g., keto to hydroxy or an olefin to aldehydeor carboxylic acid. To that extent, the choice of groups for linking tothe ligand may be varied much more widely than the choice of groups forlinking to the enzyme. In both cases, however, a wide variety ofdifferent types of functionalities have been developed, specifically forlinking various compounds to proteins and particularly enzymes.

Where a linking group is employed for bonding the ligand to the enzyme,it will be the more frequent procedure to bond the linking group to theligand to provide an active site for bonding to the enzyme. This may beachieved in a single step or may require a plurality of synthetic steps,including blocking and unblocking the active groups on the ligand, otherthan the one involved in providing the linking group. The linking groupswhich are reported hereafter are solely concerned with the bridgebonding the enzyme and the ligand.

Where a linking group is used, there will normally be from one atom to14 atoms in the chain, more usually from two atoms to 8 atoms in thechain bonding the ligand to the enzyme. Where cyclic structures areinvolved, the cyclic structure will be equated to the number of atomsproviding a similar length to the chain.

The linking group (excluding the atoms derived from the ligand andenzyme), when other than a direct bond is involved, will be of fromabout 1 to 30 atoms -- carbon, hydrogen, nitrogen, oxygen, phosphorus,and sulfur -- more usually 4 to 20 atoms.

Preferably, the linking group will normally be of from zero to 14 carbonatoms, usually from 1 to 8 carbon atoms and from 1 to 8 heteroatoms, andfrequently of from 1 to 8 carbon atoms and from 1 to 4 heteroatoms,which are oxygen, sulfur and nitrogen, more usually oxygen and nitrogen.The most frequent heterofunctionalities present in the linking group arenonoxocarbonyl or thiocarbonyl, amino, imino (oxime or imidate) diazo,or oxy.

A group of linking groups are derived from a group having anonoxocarbonyl functionality and when a second functionality is present,the second functionality may be based on a second nonoxocarbonylfunctionality, a haloalkyl, O-substituted hydroxylamine, imino, amino ordiazo. The linking group will normally have from 2 to 8 carbon atoms andfrom 1 to 4 heteroatoms which are usually oxygen and nitrogen (theheteroatoms of the ligand and enzyme are not included in the above rangeof heteroatoms). Such determination is somewhat arbitrary, so thatbetween a carbon atom of the ligand and a carbon atom of the enzyme,there may be as many as six heteroatoms. The heteroatoms may be part ofthe linking group chain or branched from the chain, e.g.,non-oxocarbonyl oxygen.

One group of linking groups will have from 2 to 6 carbon atoms, moreusually 2 to 4 carbon atoms and be an aliphatic non-oxo carbonylfunctionality. Another group of linking groups will have from 2 to 8carbon atoms and have from 1 to 2 heteroatoms, e.g., oxygen andnitrogen, in the chain, such as amino, oximino, diazo, oxy, and thelike.

The following tabulation indicates various linking groups, varying withthe functionalities present on the ligand and the enzyme. Except asindicated, the linking group satisfies one to two valences on the ligandand enzyme functional groups to which it is bound.

    ______________________________________                                        Ligand              Enzyme                                                    ______________________________________                                        amino (NH), or hydroxyl (OH)                                                                      amino(NH.sub.2), hydroxyl                                                     (OH) or mercapto (SH)                                                 ##STR41##                                                                     ##STR42##                                                                     ##STR43##                                                                     ##STR44##                                                                    P(O)(OR.sup.8)                                                                P(O)(R.sup.8)                                                                  ##STR45##                                                                     ##STR46##                                                                    C(R.sup.9).sub.2 C(R.sup.9).sub.2                                              ##STR47##                                                                     ##STR48##                                                                     ##STR49##                                                         (only primary amino)                                                                        ##STR50##                                                                     ##STR51##                                                                     ##STR52##                                                                    ZSO.sub.2                                                        ______________________________________                                    

Z- bond, hydrocarbylene of from 1 to 10 carbon atoms, more specificallyalkylene of from 1 to 6 carbon atoms, alkenylene of from 2 to 6 carbonatoms, alkynylene of from 2 to 6 carbon atoms, cycloalkylene of from 4to 10 carbon atoms and arylene of from 6 to 10 carbon atoms; oxaalkyleneof from 4 to 8 carbon atoms; and azaalkylene of from 4 to 8 carbonatoms;

R⁸ -- alkyl of from 1 to 6 carbon atoms;

R⁹ -- hydrogen or alkyl of from 1 to 3 carbon atoms;

Z or non-oxo carbonyl are preferred for bonding to hydroxyl, whilenon-oxo carbonyl, non-oxo thiocarbonyl and Z are preferred with amino.

    ______________________________________                                        Ligand            Enzyme                                                      ______________________________________                                         ##STR53##        amino (NH.sub.2), hydroxyl                                                    (OH), or mercapto (SH)                                                 NOZ                                                                           NOZCO                                                                         NO.sub.2 CZCO                                                                 CHCO                                                                          NNHZCO                                                                        NNHZCS                                                                        NOZCS                                                                         NO.sub.2 CZCS                                                                 CHCS                                                                          NOZS                                                                ##STR54##        amino (NH.sub.2), hydroxyl (OH), or mercapto (SH)                      OZCO                                                                          N(R.sup.9)ZCO                                                                 N(R.sup.9)Z                                                                   OZ                                                                            OZCS                                                                          N(R.sup.9)ZCS                                                      arylamino (Z"NH.sub.2)                                                                          methine ( CH)                                                                 amino (NH.sub.2)                                                       N                                                                  amino (NH.sub.2); hydroxyl (OH)                                                                  methine ( CH)                                                                amino (NH.sub.2)                                                       Z"N.sub.2                                                                     Z"N.sub.2                                                           ##STR55##        methine ( CH)                                                                 amino (NH.sub.2)                                                       OZ"N.sub.2                                                                    N(R.sup.9)Z"N.sub.2                                                ______________________________________                                    

Z" -- arylene of from 6 to 10 carbon atoms.

Where the enzyme is to be linked through a carboxyl group of the ligandor a linking group bonded to the ligand, either esters or amides will beprepared. The ligand may be bonded to any of the linking groups wwhichare appropriate to provide a link between the ligand and the alcohol oramine group of the enzyme to form the ester of amide group respectively.When the enzyme has an activated aromatic ring, the ligand may be bondedto an aromatic diazonium salt to provide the desired bridge.

The linking group will be selected in accordance with the followingconsiderations. The bonds formed must be stable under the conditions ofthe assay. When bonding the ligand through the linking group to theenzyme, the enzyme must retain at least a portion of its activity uponisolation. The enzyme must not prevent binding of the ligand to thereceptor. The functionalities should permit some selectivity, so thatbonding can be directed to specific groups or types of groups in boththe ligands and enzymes.

A few illustrations of how linking groups may be introduced areconsidered worthwhile. For example, if the ligand has an amino group,the amino may be bonded to form α-bromoacetamide. This product may thenbe used to form a carbon nitrogen bond to an amino acid of an enzymewhich has a free amine group, e.g., lysine.

If the ligand has a keto group, the carbonyl may be condensed directlywith an amine group of the enzyme, or the O-carboxy methyloxime may bepreapred with O-carboxymethyl hydroxylamine. A mixed anhydride, withisobutyl chloroformate is formed, which may then be used to form thecarboxamide with the amino group of the lysine.

Where a carboxyl group is present in the ligand, it may be convenient toreact the carboxy group to form the monoamide of phenylenediamine. Theresulting compound may then be diazotized to form the diazo salt whichmay be coupled with tyrosine present in the enzyme.

Another way to form the linking group would be to have an alcohol of aligand react with succinic anhydride to form the monoester. The freecarboxy group can then be activated by preparing the mixed anhydride andbe used for reaction with an amine in the enzyme.

With an amino group present on the ligand, this may be reacted withmaleic anhydride under forcing conditions to prepare the maleimide. Themaleimide may then be combined with cysteine in the enzyme to provide bya Michael's addition the 3-thiosuccinimide.

For polyfunctionalized ligands such as proteins it will usually benecessary to use special techniques to prevent the formation of enzymescoupled together which are then bonded to the ligand. Having the two ormore enzymes coupled would make inhibition difficult. Techniques can beemployed where one group of a bifunctional reagent can be madeunreactive, while the other group reacts with the enzyme or proteinligand. The other group can then be activated to carry out the secondstage of linking the protein ligand to an enzyme.

Various bifunctional reagents can be employed. For example, afunctionalized diazosulfonate can be used. One of the proteins can bebonded to the functionality and then the modified protein added to theother protein and the diazosulfonate group activated with light.

While for the most part, the enzyme may be bonded to any convenientposition of the ligand, either through a functionality naturally presentin the ligand or one introduced synthetically, there are preferredmethods of bonding the enzyme to the ligand. First, it should berecognized that the ligand of the enzyme-bound-ligand need not have anybiological activity. One is primarily concerned in not disturbing thegeometry and polar site relationships of a substantial portion of theligand molecule. Where the ligand is a hapten, the enzyme will thereforenormally be bonded at the same site as was employed for attachment tothe protein in the preparation of the antigen. Where the ligand is anintact antigen, several sites may be employed for attachment to one ormore enzyme molecules with the obvious limitation that the number ofenzyme molecules must not be so great as to prevent binding to theantibody. Where the ligand has a natural receptor other than anantibody, the point(s) of attachment will also be determined primarilyby the necessity to preserve strong binding to the receptor.

Furthermore, if one is attempting to assay one of a variety of moleculeswhich are quite similar, for example steroids, but differing in theirsubstituents at the 17 position, one would choose to mark the moleculewith the enzyme at a site distant from the distinguishing functionality.Following the steroid analogy, it would frequently be preferable to bondat the 3 position, rather than at the 17 position, since the distinctiveportion of the molecule is usually at the 17 position. For the mostpart, the 3 position is either an alcohol or a ketone, the ketonenormally being associated with aliphatic unsaturation. Also, the 6position is a useful site.

The same or similar consideration will be present with other ligands.For example, with a polypeptide, which has a natural receptor site, onewould preferably bond away from the receptor site.

The number of ligands which may be bonded to the enzyme will be limitedby the number of available sites for bonding to the enzyme. In mostcases this will be the amino groups which are present, but as alreadyindicated, carboxyl, hydroxyl, thiol and activated aromatic rings, e.g.,phenolic, are also useful sites.

Various factors will affect the number of ligands which is optimum for aspecific enzyme and a specific ligand. Of prime consideration is thenumber required for obtaining the desired degree of inactivation whenreceptor is bound to the enzyme-bound-ligand. The number required willvary with the mode of attachment and the conditions for attachment ofthe ligand to the enzyme. Except under special circumstances, e.g.,affinity labelling, there will usually be differences in degree ofinactivation, as to each site to which the receptor is bound to theenzyme through a ligand. In addition, there may be cumulative effects,with an increase in the number of receptors bound to the enzyme throughligand.

Other considerations as to the number of ligands per enzyme will be theeffect of the increasing number of ligands on: solubility of theenzyme-bound-ligand; activity of the enzyme-bound-ligand in the absenceof receptor; and the sensitivity of the assay. Therefore, the choice ofthe number of ligands bonded to the enzyme is usually empiricallydetermined, based on the effect of varying the number of ligands on theenzyme has on the assay.

With small enzymes, e.g., lysozyme, those that have molecular weights inthe range of 10,000 to 30,000 from 2 to 10 ligands can be sufficient.With larger enzymes, e.g., malate dehydrogenase, of molecular weight inthe range of 30,000 to 150,000, 2 to 30 ligands can be sufficient. Formalate dehydrogenase 2 to 22 ligands on the average will be employed. Asfew ligands as possible should be bonded to the enzyme to achieve thedesired degree of inhibition. Desireably, the number of ligands perenzyme should be in the range of 1 to 20, more preferably 1 to 12.

As already indicated, because of the diversity of enzymes which can beused for the assay and the variety of functionalities in the enzymeavailable for attachment, and the varying activities of thefunctionalities for being bonded to the ligand as well as their relativeposition to the active site of the enzymes, different numbers of ligandswill be necessary for obtaining the desired degree of inhibition, whenthe enzyme-bound-ligand is bonded to antibody. Furthermore, the desireddegree of inhibition may vary, depending on the sensitivity required foran asssay for a particular ligand.

It is found, for the most part, that increasing the average number ofligands increases the amount of inhibition, up to a degree ofsubstitution, where further substitution does not provide a significantincrease in inhibition. Therefore, by varying the conditions for thereaction between the modified ligand (ligand and linking group) and theenzyme, varying degrees of substitution can be achieved. The time forthe reaction, the mole ratio of ligand to enzyme and the like can bevaried. Also, the reactive functionality on the linking group can bevaried to change the number and sites for substitution. One can thenempirically determine the number of ligands required for the desireddegree of inhibition.

It should also be noted that in referring to inhibition of an enzyme,the substrate for the enzyme plays a role. Different degrees ofinhibition may be achieved with different substrates. Thus, not only canone obtain varying degrees of inhibition by varying the number ofligands bonded to the enzyme, and the sites to which the ligands arebonded, but also, with some enzymes, by varying the substrate for theenzyme.

It is also found that with increasing substitution of the enzyme byligand, there can be reduction in enzyme activity. The turnover numberdiminishes and there is a concomitant increase in the Michaelisconstant. The decrease in turnover number with increasing substitutionwill vary with the enzyme. By employing enzymes which have a highinitial activity, a loss of as much as 75 percent of initial activitycan be tolerated.

(Turnover number is the number of substrate molecules transformed perunit time per enzyme molecule. Lehninger, Biochemistry, WorthPublishers, New York, 1970. Since the turnover number is reported atvarying temperatures and on varying bases, e.g., weight of protein as anindication of number of enzymes or change in a spectrophotometric valueas an indication of number of substrate molecules, there is at thepresent no simple conparison between the turnover number of differentenzymes. Therefore, no minimum numerical turnover number for preferredenzymes can be given.)

Also, the ligand will be attached to the enzyme by a relatively shortchain, usually of the order of 1.5 to about 20 A, more usually about 3to 10 A.

Enzyme Assay

Turning now to a consideration of the determination of the amount ofactive enzyme, assaying for enzymatic activity is well established for awide variety of enzymes. A wide diversity of media, conditions andsubstrates have been determined for measuring enzymatic activity. See,for example, Bergmeyer, Methods for Enzymatic Analysis, Academic Press,New York, 1965. Since there are differences, not only between assays fordifferent enzymes, but even in the variety of assays for a particularenzyme, no general description of the assay techniques can be given.

Receptor

In the subject invention, for the most part, the receptors will bemacromolecules which have sites which recognize specific structures. Therecognition of the specific structures will be based on van der Waalsforces, which provide a specific spatial environment which maximizes thevan der Waals forces; dipole interactions, either by permanent orinduced dipoles; hydrogen and ionic bonding; coordinate covalentbonding; and hydrophobic bonding. For a detailed discussion ofmechanisms by which receptors bind ligands, see Goldstein, et al.,Principles of Drug Action, Harper and Rowe, New York, 1968.

The macromolecules of greatest interest are proteins and nucleic acidswhich are found in cell membranes, blood, and other biological fluids.These compounds include enzymes, antibodies, ribonucleic acid (RNA) anddeoxyribonucleic acid (DNA) and natural receptors.

The most convenient group of proteins for use in the subject inventionare antibodies. These materials are conveniently used in the analysis ofthe category of ligands referred to as haptens. Antibodies are producedby introducing an immunogenic substance into the bloodstream of a livinganimal. The response to the introduction of the immunogenic substancefor anitgen is the production of antibodies which act to coat theantigen and detoxify it or precipitate it from solution. The proteinforms a coat which is geometrically arranged so as to have the antigenfit the spatial arrangement of the protein. This may be analogised to alock and key. The interaction is normally reversible, in that theantigen is subject to displacement or removal by various means withoutdestruction of the receptor site.

There are many materials which are antigens and will produce animmunogenic response by being introduced into the bloodstream of avertebrate. However, a number of materials of interest are not antigens,but are haptens, and in that situation, an extra step in preparing theantibody is required. This method of preparing antibodies with materialsother than antigens is well known and may be found in Microbiology,Hoeber Medical Division, Harper and Rowe, 1969. See also, Landsteiner,Specificity of Serological Reactions, Dover Publications, N.Y. 1962;Kabat, et al., Experimental Immunochemistry, Charles C. Thomas,Springfield, Illinois, 1967; and Williams, et al, Methods in Immunologyand Immunochemistry, Vol. I, Academic Press, New York, 1967.

The material which is to be assayed is bonded to a protein by anyconvenient means and the modified protein introduced into the bloodstream. The same type of bonding groups used with the enzyme attachmentto the ligand may be employed. The antibodies which form will includegroups of antibodies which are shaped to fit the foreign moiety bondedto the protein. Therefore, antibodies are obtained which are specific tothe compound or moiety bonded to the protein. By careful separationtechniques, the antibodies primarily concerned with the moiety inquestion, can be concentrated so as to provide an antibody compositionwhich is primarily related to the specific moiety which was bonded tothe protein.

To illustrate this method, para-aminobenzene arsonate is diazotized toform the diazo salt. By combining the diazo salt with rabbit globulin,the rabbit globulin may be labeled with para-azobenzene arsonate. Byintroducing this composition into the blood stream of an animal otherthan a rabbit, for example a sheep, antibodies can be formed which willhave a spatial arrangement which accepts solely the azobenzene arsonate.

In addition to antibodies, there are a number of naturally occurringreceptors which are specific to compounds of biological interest.Compounds for which receptors are naturally occurring include thyroxine,corticosterone, cortisone, 11-desoxycortisol, 11-hydroxyprogesterone,estrogen, insulin and angiotensin. See, for example, Vonderhaar et al,Biochem. Biophysics Acta., 176, 626 (1969). All of these ligands havebeen studied and reported upon in the literature in connection withstudies on their binding with specific receptors.

                                      Table I                                     __________________________________________________________________________    Ligand   Receptor for Ligand Reference                                                                     Ligand Structure                                 __________________________________________________________________________    Thyroxin Thyroxin Binding Globulin (TBG) Thyroxin Binding Prealbum (TBA)               B.E.P. Murphy, C.J.J. Pattee, J. Clin. Endocr., 24, 187                                            ##STR56##                                                                    Thyroxine                                        Corticosterone                                                                         Protein From Brain Cell Nuclei, B. McEwen, L. Plapinger Nat.                  226, 263 (1970)                                                                                    ##STR57##                                                                    Corticosterone                                   Cortisol (R = OH,H) Cortisone (RO) 11-desoxycort- isol (R                              B.E. Murphy, J. Clin. Endocr., 28, 343 (1968), 27, 973 (1967)                 Corticosteroid Binding Globulin (Transcortin)                                                      ##STR58##                                                                    Cortisone                                        Estradiol                                                                              Receptor Site for Estrogen From Uterus, BBA, 176, 626                                              ##STR59##                                                                    Estradiol                                        Insulin  C.R. Morgan, W.M. Holland, III                                                                    *see below                                                Diabetes, 1966                                                        ##STR60##                                                                     Angiotensin II                                                                        L. B. Page, E. Haber, A. Y. Kimura                                                                *see below                                                A. Pernode, J. Clin. End. 28, 200                                             (1969)                                                               __________________________________________________________________________     *Asp-Arg-Val-Tyr-Ileu-His-Pro-Phe                                        

Generally, the experience obtained in bonding a specific hapten to aspecific enzyme can be used in bonding other haptens to the same enzyme.This is truer the more similar the haptens. Therefore, with drugs havingsimilar solubilities one will ordinarily expect to obtain similarresults with different but similar haptens, when bonding the haptens tothe enzyme with the same linking functionality. It has therefore beenfound synthetically convenient to employ O³ -carboxymethylmorphine as aprototype to evaluate a wide variety of enzymes when bonded to acarboxyl group by means of a mixed anhydride. The information thusobtained can be readily extrapolated to what one would expect frombonding other similar drugs in an analogous manner to the same enzyme.

EXPERIMENTAL

The following examples are offered by way of illustration and not by wayof limitation.

(All temperatures are recorded in Centigrade).

INDEX

A. preparation of morphine antibodies to O³ -carboxymethyl morphineconjugate and binding to support.

1. Morphine

1.1 O³ -Carboxymethyl morphine conjugate to amylase

1.2 O³ -Carboxymethyl morphine conjugate to horse radish peroxidase.

1.3 O³ -Carboxymethyl morphine conjugate to lysozyme.

1.4 O³ -Carboxymethyl morphine conjugate to malate dehydrogenase.

1.5 O³ -Carboxymethyl morphine conjugate to malate dehydrogenase withvarying ratios of morphine to malate dehydrogenase.

1.6 O³ -Carboxymethyl morphine conjugate to lactate dehydrogenase.

1.7 O³ -Carboxymethyl morphine conjugate to glyoxylate reductase.

1.8 O³ -(α-Isopropyl)carboxymethyl morphine conjugate to malatedehydrogenase.

1.9 O³ -Carboxymethyl morphine conjugate to glucose 6-phosphatedehydrogenase.

1.10 O³ -Imidoylmethyl morphine conjugate to lysozyme.

1.11 O³ -Imidoylmethyl morphine conjugate to glucose 6-phosphatedehydrogenase.

2. Methadone

2.1 6-Keto-7,7,-diphenyl-9-dimethylaminodecanoic acid conjugate tolysozyme.

3. Meperidine

3.1 4-Carbethoxy-1-carboxymethyl-4-phenylpiperidine conjugate tolysozyme.

4. Amphetamine

4.1 N-Carboxymethyl amphetamine conjugate to lysozyme.

5. Barbiturates

5.1 N-Carboxymethyl phenobarbital conjugate to lysozyme.

5.2 5-(γ-Crotonic acid)-5-(2'-pentyl)-barbituric acid conjugate tolysozyme.

5.3 N-Carboxymethyl glutethimide.

5.4 N-(4-Carboxybutyl)phenobarbital conjugate to lysozyme.

5.5 5-(γ-Crotonic acid)-5-(2'-pentyl)barbituric acid conjugate tolysozyme.

6. Cocaine

6.1 Ecgonine conjugate to lysozyme.

6.2 p-Diazobenzoyl ecgonine conjugate to lysozyme.

7. Insulin

7.1 p-Diazobenzamide modified insulin conjugate to malate dehydrogenase.

8. Steroids

8.1 Testosterone-3-carboxymethyloxime conjugate to malate dehydrogenase.

8.2 3-(O-Carboxymethyl)estradiol conjugate to malate dehydrogenase.

EXAMPLE A Preparation of Morphine Antibodies and Binding to Support

1. Morphine (900 mg.) was dried for 4 hours at 50° C., 0.01 mm. Hg. Thedried morphine was dissolved in 18 ml. of abs. ethanol and 125 mg.sodium hydroxide was added, followed by the addition of 350 mg. drysodium chloroacetate. After purging with nitrogen, the solution wasstirred and refluxed for four hours. The hot solution was treated with3.8 ml. ethanolic hydrogen chloride (0.85 M) and then filtered whilestill warm. On cooling overnight, a precipitate (272 mg.) formed whichwas collected and recrystallized from ethanol/water. On addition ofether to the original filtrate an additional precipitate was obtainedwhich was also recrystallized from ethanol/water. Total yield 600 mg(55%). On heating this product to 75° C in vacuo there was a weight losscorresponding to 0.48 molecule of ethanol or 1.15 molecule of water. Thedried compound decomposes at 190°-220° (depends on rate of heating).

Anal: C₁₉ H₂₁ NO₅ ; % Theor: C,66.45; H,6.16; N,4.08: % Found: C,65.87;H,6.98; N, 4.09,4.07: NMR(C₅ D₅ N) 2.44 ppm (-CH₃), 5.08 ppm (-CH₂-COO).

2. Carboxymethyl morphine (240 mg) suspended in 8 ml dry dimethylformamide (DMF) was cooled to -15° C and treated with 84 μl isobutylchloroformate. The solid dissolved while stirring for 30 minutes at -15°C. Bovine serum albumin (BSA) (400 mg) dissolved in 56 ml watercontaining 2.6 g sodium bicarbonate was added to this solution and themixture was kept at 0° C overnight. It was then dialyzed againstdistilled water with four changes of water (dialysis 1:80) andlyophilized to give 350 mg of conjugate.

Hapten concentration on the protein:

    ______________________________________                                        MW.sub.CMM = 327  ε.sup.280 .sub.BSA = 41600                          MW.sub.BSA = 64 400                                                                             ε.sup.280 .sub.CMM = 1070                           ______________________________________                                    

The ultraviolet spectrum was measured at 280 nm. in a 1 cm.cell; d=0.59when the concentration was 0.287 g/l in water. The degree of conjugationcan be determined from the above data and the formula: ##EQU1## where X= number of haptens per molecule, W = weight of protein conjugate perliter and MW is the molecular weight where CMM refers to the haptencarboxymethylmorphine, and BSA refers to the protein.

    X = 46.6 haptens/molecule

3. Antisera may be obtained as follows: The antigen (hapten coupled toan appropriate protein, see above example) is made up in a salinesolution (9 g./liter) at a 2 mg./ml. concentration. Per b 1.0 ml.aliquot of the above solution, introduced, there is introducedsimultaneously 2 ml. of Complete Freund's Adjuvant in homogenized formby means of a two-way needle. For subcutaneous injections, approximately0.3 ml. (antigen + Freund's solution) is injected per site and forintraperitoneal injections, approximately 0.4 ml. is injected. The totaldosage is about 4.0 ml. per rabbit.

After 3 to 4 weeks, a booster shot is given intramuscularly consistingof 0.5 ml. of the above saline solution and 0.5 ml. of Complete Freund'sAdjuvant. A period of 5 to 7 days is allowed to pass and the rabbit isbled by heart puncture.

When the desired amount of blood is collected, the blood is allowed toclot and the clot removed. The remaining solution is then centrifuged at2,000 R.P.M. for 10 minutes. The serum is collected free of loose redcells.

An equal volume of saturated ammonium sulfate solution is added to theserum dropwise with stirring at 4° C. After standing for 1 hour at thattemperature, the solution is centrifuged at 10,000 R.P.M. for 15 minutesand the supernatant removed. The residue is suspended in as small avolume as possible of 1 X BB (borate buffer, see below for description),transferred to a dialysis bag and dialyzed overnight against BB, pH 8.0.The residue in the dialysis bag is then isolated and frozen.

(To make borate buffer, dissolve 24.6 g. boric acid in water, adjust thepH with sodium hydroxide to a pH 7.9-8.0, add 0.1 g. of sodium azide and0.01 g. of ethylmercurithiosalicylate and bring the total volume to oneliter).

4. Into 20 ml. of dimethyl formamide is introduced 400 mg.aminoethyl-Bio-Gel P-300 and 300 mg. of carboxymethyl morphine (SeeExample A-1) and 1 g. sodium bicarbonate is added. After stirring thesuspension for two days at 4° C., the suspension is filtered, theresidue is washed with water until the washings are neutral, and thenthe residue is dried in vacuum.

The resulting product is then suspended in 20 ml. rabbit serumcontaining morphine antibodies and is stirred for 4 hours at 4° C.Filtration gives a residue which is resuspended in 5 ml. phthalatebuffer, pH 3.8 (0.1 M) and is stirred for 2 hours. The gel is separatedby centrifugation and the supernatant liquid is dialyzed againstphosphate buffer, pH 7.4 (0.1 M) to give a buffered solution ofsubstantially pure antibodies.

5. Into a flask were combined 5 ml. Sepharose 4B suspension, 5 ml.water, and 5 ml. 100 mg./ml. CNBr solution and the pH adjusted to 11.5with 4N NaOH. While stirring the mixture, the pH was metered andmaintained at 11-11.5 with 4N NaOH until no further change in pH wasnoted. The mixture was filtered and washed with 100 ml. of 0.1 Mcarbonate buffer, pH 9.0.

Morphine antibodies were pretreated by passing 3 ml. of the antibodiesthrough a 10 × 0.9 cm. column of Sephadex G-25 equilibrated against 0.1M carbonate, pH 9.0. The eluate was 4 ml.

Sepharose prepared above was suspended in 5 ml of carbonate buffer(cold) and added to the morphine antibody solution. the mixture wasstirred for 36 hours at 4° C, filtered and washed with 100 ml of 0.1 Mcarbonate buffer, pH 9. Ultraviolet analysis of the washings showed 65%of the protein bonded to the Sepharose.

6. Receptor sites can be determined as follows: Enzyme activity of asolution containing enzyme-bound-ligand is determined according tonormal methods. Antibody for the ligand is added until there is nofurther change in enzyme activity by the further addition of antibody.This determines the residual activity with substantially all of theenzyme-bound-ligand bound to antibody.

A second solution is provided of antibodies and enzyme-bound-ligand isadded incrementally with the enzyme activity monitored after eachaddition. The enzyme activity is plotted against the amount ofenzyme-bound-ligand added. In effect, antibody is being titrated withenzyme-bound-ligand. At some point, substantially all the receptor siteswill be filled and one will obtain a straight-line relationship betweenthe increase in enzyme activity and the amount of enzyme-bound-ligandadded. By extrapolating the straight line portion to the abscissa, oneobtains the effective number of antibody sites in inhibitingenzyme-bound-ligand. By increasing the effective number by the percentof residual activity, a close approximation of the absolute number ofantibody sites is obtained.

EXAMPLE 1.1

A. To a solution of 100 mg. of amylase in 15 ml. of water containing 600mg. of sodium bicarbonate was added 4.6 × 10⁻² M of the mixed anhydrideof morphine (prepared as described in Example A.2) in 1 ml. DMF at0°-5°. The solution was stirred at 0° for 18 hours, transferred to adialysis bag and dialyzed against water for 2 days at 0°. The residuewas lyophylized to a white solid.

The activity of the morphine modified amylase was determined as follows:Morphine modified amylase was mixed with a suspension of dyed amylose at37°. After an arbitrary time, the reaction was quenched, centrifuged andthe supernatant liquid transferred to a cuvette and the optical densitymeasured. This was repeated a number of times so that the opticaldensity could be plotted versus time to give a linear graph. The slopeof the line indicated the enzyme activity. It was found that themodification of the amylase with morphine had little, if any, effect onthe enzymatic activity of the starting amylase.

B. A measured amount of morphine antiserum (concentration of activesites equals 2 × 10⁻⁷ M per liter) was added to a 6.4 × 10⁻⁸ M./lcarboxymethylmorphine modified amylase solution. The amylase activitytest was then carried out at 37° for a total time of 20 minutes. Thefollowing table indicates the results:

    ______________________________________                                                  Volume of carboxy-                                                                           Volume of                                                      methylmorphine modi-                                                                         antibody   Optical                                   Determination                                                                           fied amylase, μl.                                                                         solution, ml.                                                                            Density                                   ______________________________________                                        1         100            0.0        0.910                                     2         100            0.020      0.720                                     3         100            0.050      0.560                                     4         100            0.100      0.450                                     5         100            0.150      0.390                                     ______________________________________                                    

The above table shows that with increasing concentration of antibody,there is decreasing activity of the carboxymethylmorphine modifiedamylase.

C. The effect of addition of codeine was determined by running onesample containing antibody and carboxymethylmorphine in the absence ofcodeine and one sample in the presence of codeine.

With 0.2 ml. of a solution containing antibodycarboxymethylmorphinemodified enzyme complex employed in the amylase determination, theresulting optical density was 0.450. However, when 0.030 ml. of a 10⁻⁷ Mof codeine solution was added to the same amount of antibody-enzymecomplex, the resulting optical density in the amylase assay was 0.580.In this manner, a solution containing 10⁻⁷ M codeine could be assayedwhere only 30 microliters were available. This is not intended toindicate, however, that this is the minimum concentration required, butrather only that it can be successfully employed. The above method couldalso be used with morphine, morphine glucuronide, as well as other closestructural analogs of morphine and codeine.

EXAMPLE 1.2

(1) To 10 mg (2.5 × 10⁻⁷ M) horseradish peroxidase (3400 I.U./mg) and 20mg NaHCO₃ in 1 ml water was added 0.09 ml (9 × 10 ⁻⁶ M) of a 100μmole/ml solution of carboxymethylmorphine mixed anhydride (see below)in dimethyl formamide (DMF) and the mixture was allowed to standovernight at 4° C. The solution was then passed through a 1 × 10 cmcolumn packed with Sephadex G-25 and the effluent used forperoxidase-bound-morphine in assaying for morphine.

(2) The isobutylchloroformate O³ -carboxymethylmorphine mixed anhydridewas prepared as 100 μmole/ml DMF solution. Five mg of horseradishperoxidase (HRP) (1.5 × 10⁻⁶ mole of lysine residues) was dissolved in0.5 ml of water. The solution was cooled to 4° C and the pH was adjustedto 9.5 with dilute NaOH solution. Ninety μl (9 × 10⁻⁶ mole) of the mixedanhydride was added in 10 μl portions while the pH was maintained at 9 -10 by intermittent addition of NaOH. The mixture was stirred for onehour and dialyzed exhaustively against water.

EXAMPLE 1.3

A first solution (solution A) was prepared by dissolving 100 mg oflysozyme (6.9 × 10⁻⁶ mole) in 10 ml of water and adding sodium carbonateuntil a pH of 8.0 was obtained.

A second solution was prepared by suspending 34.3 mg (1 × 10⁻⁴ M) ofcarboxymethylmorphine in 2 ml of anhydrous DMF. After cooling to -15° C,13.1 μl of isobutyl chloroformate (1 × 10⁻⁴ M) was added to form a mixedanhydride. The solution was stirred at -15° C for about 30 minutes atwhich time the solids had dissolved.

Solution A was cooled in an ice bath and the above morphine mixedanhydride added. After storing at 4° C for 14 hours, the solution wasdialyzed against distilled water for 4 days (2,000 ml of water replacedtwice daily).

The protein was lyophylized and the residue dissolved in 10 ml of 0.128Msodium phosphate, pH 7.15. The product mixture was fractionated on aweak acid cation exchange resin column (Biorex 70), eluting at a rate of1 ml per minute with a 400 ml linear gradient ranging from 0.128 to0.400M sodium phosphate, pH 7.15. The eluent was collected in 2 mlfractions. The chromatography was continuously monitored by measuringultra-violet absorption at 280 nm. Five fractions were obtained. Thelysozyme activity was measured according to the following technique.

A 0.35 mg/ml suspension of dried bacteria, Micrococcus lysodeikticus, in0.05M sodium phosphate, pH 7.0 is prepared. To 2.85 ml of the suspensioncontained in a 3 ml cuvette is added 0.10 ml of 8.76% (W/V) (g/l.)sodium chloride solution and 0.025 ml enzyme solution (7.5 × 10⁻⁶ g ofprotein). The contents are mixed and placed in a spectrophotometer setat 436 nm at 30°. The rate of decrease of optical density with time isrecorded and the rate expressed as optical density units per minute per7.5 × 10⁻⁶ g of protein. The following table indicates the fractions,the milligrams of protein per milliliter, and the rate of reaction.

    ______________________________________                                                                  rate OD/min/                                                                  7.5 × 10.sup.-6 g                             Fractions  mg protein/ml  protein                                             ______________________________________                                        a          0.15           0.027                                               b          0.18           0.033                                               c          0.26           0.040                                               d          0.28           0.047                                               e          0.31           0.043                                               Lysozyme   0.30           0.070                                               ______________________________________                                    

Except where otherwise indicated, the following is the procedure forassaying, when the ligand is conjugated to lysozyme. A buffer solutionis prepared of Tris-Maleate, 0.025 M, pH 6.0, by dissolving 3.03 g ofTris and 2.9 g of maleic acid in 800 ml of distilled water. Afteradjusting the pH to 6 with 1N sodium hydroxide, the solution is dilutedto a final volume of 1 liter. A 0.1 weight percent bovine serum albumin(BSA) solution in the above buffer was prepared by diluting 1 g of BSAin 1 l of the buffer. A substrate solution was prepared by suspending 30mg of M. lysodeikticus (also referred to as M. luteus) (Miles,lyophilized) in 50 ml of the above buffer and is prepared 12 hoursbefore use and stored at 4° in a plastic container. The stock solutionof the carboxymethylmorphine conjugated to lysozyme is diluted with theBSA solution so as to obtain a reagent solution having a rate of lysisof about 0.210 ± 0.020 OD/min.

The active lysozyme content of the working solution is determined bymeasuring at 436 nm the rate of bacterial lysis at 30°. The solution isprepared by mixing 0.200 ml of bacterial solution, 0.020 ml of the BSAsolution, 0.080 ml of synthetic urine, and 0.500 ml of the enzymesolution.

The antibody solution employs a 0.025 M Tris-Maleate buffer at pH 7.4and is employed in a sufficient amount in the assay to inhibit 92 - 96%of the enzyme activity of the stock enzyme solution.

In carrying out the assay 0.2 ml of the bacterial suspension is pipettedinto a flask to which is added 20 μl of the antibody solution. A urinesample is then introduced carefully, and 0.5 ml of the enzyme solutionadded. The reaction mixture is then aspirated into the spectrometer andthe decrease in optical density measured at 436 nm for 10 seconds (anytime interval from 10-60 seconds may be used) at 30°. The concentrationof morphine present in the urine sample may then be determined from astandard curve.

EXAMPLE 1.4

Porcine heart malate dehydrogenase (MDH) (0.3 cc, 1 × 10⁻⁷ moles mDH) asa 10 mg/ml suspension in 3.2 M ammonium sulfate, was centrifuged. Thepellet was dissolved in 0.2 cc of water and dialyzed against 125 cc ofwater at 3° for 3.5 hours with one water change. The dialysate wasdiluted to 1 cc with a solution 0.15 M sodium phosphate and 0.075 Msodium carbonate at pH 9.0. Seventy-four μl of the mixed anhydridesolution prepared as described previously was added in 5 μl incrementswith stirring at 0°. During the addition, the pH was maintained between8.8 and 9.0. After the addition was complete, the solution was adjustedto pH 9.5 and stirred for 1.5 hours at room temperature. The solutionwas dialyzed for 24 hours against 125 ml of 0.05 M phosphate-0.05 Mcarbonate buffer, pH 9.3 at 3° with three buffer changes. The malatedehydrogenase was found to have about 27 morphine groups per enzymemolecule.

EXAMPLE 1.5

The following was a study concerning the effect of increasingsubstitution by carboxymethylmorphine with porcine heart malatedehydrogenase.

Malate dehydrogenase (4.0 ml, 40.0 mg, Calbiochem Lot 101089) wascentrifuged (17,500 rpm, 20 minutes). The resulting pellet was dissolvedin 1 ml of distilled water and dialyzed against 250 ml of 0.01 Mphosphate buffer, pH 7.5 at 3° with 2 × 250 ml buffer changes in 4hours. The resulting dialysate was diluted to 5 ml with 0.15 M phosphate-- 0.075 M carbonate, pH 9.0, to give a solution approximately 0.1 Mphosphate -- 0.05 M carbonate. The solution was divided into two 2.5 mlportions.

One of the portions was cooled to approximately 0° in an ice bath, and a0.1 M solution of radioactive O³ -carboxymethylmorphine (2.43 × 10⁵counts per minute per μmole) was added with rapid stirring in 4 - 5 μlincrements. Four aliquots of from 0.4 - 0.5 ml were withdrawn atappropriate times, the additions and withdrawals being carried out atthe following schedule, while maintaining the pH of the solution between8.8 and 9.0. The samples were withdrawn when the pH indicated reactionhad occurred.

The total additions prior to each sample withdrawal were: (1) 15 μl; (2)25 μl; (3) 19.8 μl; (4) 34 μl; (5) 41.5 μl. Five minutes after the lastaliquot was added, the sample was withdrawn.

The pipettes employed to withdraw the 0.5 ml samples were each rinsedwith 0.25 ml 0.05 M phosphate -- 0.05 M carbonate buffer, pH 9.5. Theoriginal solutions and rinses were quantitatively transferred todialysis sacks with proper rinsing and dialyzed for about 48 hours at 3°against the same phosphatecarbonate buffer with 5 × 250 ml bufferchanges.

The samples were then quantitatively removed from the dialysis bags,using the same buffer rinses and diluted to 2 ml with the same buffer.Sample number four had a small amount of precipitate, while samplenumber five had a large amount of precipitate. Both samples werecentrifuged, (17,500 rpm, 20 minutes). The pellet from sample four waswahsed with buffer, twice with small amounts of water, followed by analcohol wash, and then dried in a nitrogen stream, to yield 0.2 mg.Efforts to dissolve the precipitate from sample number five were notsatisfactory.

From each of the samples, a 0.5 ml aliquot was withdrawn, (thesupernatant of sample four was employed) and each added to 10 ml ofscintillation fluid and counted. Sample number one had a count of11,350; sample number two 33,700, sample number three 55,200; and samplenumber four 80,100.

This calculates out to 3.4, 10.4, 17.3, and 27.8 morphine molecules perenzyme for the first to fourth samples. Because of the substantialinsolubility of the fifth sample, no data were obtained for it.

The second sample of enzyme was treated substantially in the same mannerdescribed above to give an additional four samples ofcarboxymethylmorphine substituted malate dehydrogenase, labeled with3.2, 6.1, 13.2, and 17.3 morphine.

The samples were then assayed as follows: an assay solution was preparedby combining 0.92 ml of 0.5 M phosphate buffer, 50 μl of 7 mMoxaloacetate in phosphate buffer, 20 μl of 14 mM NADH and 1 μl of 3.67 ×10⁻⁵ in binding sites of antibody (binding sites determined by FRAT®, anESR technique supplied by Syva Corp.) which is a large excess over themorphine present. To this solution was added 10 - 20 μl of thecarboxymethylmorphine modified malate dehydrogenase, which had beendiluted 1,000 fold with 1 M potassium monoacid phosphate solution.

The rate of the reaction can be followed by metering the change inoptical density, at 340 nm. Approximately 30 seconds is required to mixthe various reagents and the reading is then taken for the second or thethird minute, depending on which gave the faster rate. Sincethermodynamic equilibrium is not achieved within the time the readingsare taken, the rate is changing with time. However, by repeating thesame procedure before and after the addition of antibody, relativepercents inhibition can be obtained for the time limit which isdesirable for a commercial assay.

    ______________________________________                                                  Activity    Activity                                                          without Ab  with Ab     % Inhi-                                     Sample No.                                                                              OD/min.     OD/min.     bition                                      ______________________________________                                        1 a       0.117       0.087       26                                          b         0.141       0.116       18                                          2 a       0.078       0.039, 0.037                                                                              53                                          b         0.125       0.087       30                                          3 a       0.071       0.017, 0.015                                                                              78                                          b         0.125       0.048       62                                          4 a       0.066       0.008, 0.009                                                                              86                                          b         0.143       0.033       77                                          ______________________________________                                    

The above data demonstrate two facts for malate dehydrogenase: (1) withincreasing substitution there is decreasing initial activity; and (2)with increasing substitution there is increased inhibitability for theenzyme. Therefore, when randomly substituting malate dehydrogenase, onecompromises between the percent inhibition to obtain an acceptable assayand initial activity of the enzyme.

EXAMPLE 1.6

Beef heart lactate dehydrogenase (LDH) (molecular weight 142,000; 50IU/mg) was employed as a 34 mg/ml suspension in ammonium sulfatesolution. A 0.47 ml aliquot of the suspension was centrifuged and thepellet containing 8 mg of LDH was dissolved in 0.5 ml 0.01 M phosphatebuffer, pH 7.5 and dialyzed, against the buffer. The dialysate solutionwas diluted to 20 ml with 0.15 M phosphate -- 0.05 M carbonate buffer,pH 9.0 and the solution divided in half.

Each half containing 4 mg of enzyme (5.64 × 10⁻⁸ mole) was cooled to 4°and radioactive O³ -carboxymethylmorphine mixed anhydride with isobutylcarbonate (100 μmole/ml dimethyl formamide) was added. To the firstsample 17 μl (1.7 × 10⁻⁶ mole) was employed, while with the secondsample 34 μl (3.4 10⁻⁶ mole) was employed. During the reaction, the pHwas maintained at 9 by addition of dilute NaOH.

After 30 minutes, the solutions were dialyzed exhaustively against 0.05M phosphate -- 0.05 carbonate solution, pH 9.3. The dialysate solutionswere then diluted to 2 ml.

Scintillation counting of the samples showed that sample number 1 had anaverage of about one carboxymethylmorphine per enzyme molecule, andsample number two had an average of 2.5 carboxymethylmorphines perenzyme molecule.

The products were then assayed in the conventional manner employingpyruvic acid, sodium salt, and NADH. (See Worthington enzyme catalog fordetails.)

The results are as follows. With 3 μl of the first enzyme sample, theactivity was found to be 0.141 OD/min. When 10 μl of 8.3 × 10⁻⁵ Mantibody (based on binding sites) was added, the rate was 0.110 OD/min.When, in addition to antibody, 30 μl of 3 × 10⁻⁴ M codeine was added,the rate rose to 0.132 OD/min.

With the second sample 3 μl of the enzyme solution had an activity of0.142 OD/min. The addition of 10 μl of 8.3 × 10⁻⁵ M antibody reduced therate to 0.095 OD/min. When, in addition to the antibody, 30 μl of 3 ×10⁻⁴ M codeine, the rate rose to 0.125 OD/min.

EXAMPLE 1.7

Spinach leaf glyoxylate reductase (GR) was obtained as a suspension inammonium sulfate (5 mg/ml) solution. An aliquot of the suspension wascentrifuged and the pellet containing 5 mg of GR was dissolved in 0.5 ml0.01 M phosphate, pH 7.5 and dialyzed against the phosphate buffer. Thesolution was then diluted to 2 ml with 0.15 M phosphate -- 0.05 Mcarbonate buffer, pH 9.0 and divided in half. Each half, containing 2.5mg of GR (2.46 × 10⁻⁸ mole) was cooled to 4° and radioactive O³-carboxymethylmorphine mixed anhydride with isobutyl carbonate (100μmole/ml DMF) added: to the first aliquot, 8 μl (8 × 10⁻⁷ mole); to thesecond aliquot 16 μl (1.6 × 10⁻⁶ mole). The pH was maintained at 9 byaddition of dilute sodium hydroxide. After 30 minutes, solutions weredialyzed exhaustively against 0.05 M phosphate-0.05 M carbonate, pH 9.3.The dialysate solutions were then diluted to 2 ml.

Scintillation counting indicated 2.2 carboxymethylmorphines per enzymein the first sample, and 5.5 carboxymethylmorphines for the secondsample.

The assays were carried out in the same manner as that employed forlactate dehydrogenase. The enzyme samples were diluted ten-fold beforeuse.

With the first sample, 5 μl had an activity of 0.098 OD/min. whichdiminished to 0.093 OD/min. when 10 μl of 8.3 × 10⁻³ M of antibody wasadded. With the second sample 5 μl had an activity of 0.85 OD/min.,which diminished to 0.070 OD/min. when 10 μl of antibody solution wasadded. When 30 μl of a 3 × 10⁻⁴ M codeine solution was added to themixture containing antibody, the rate was 0.075 OD/min.

EXAMPLE 1.8

A. A mixture of 9.45 g (30.2 mmoles) morphine and 1.22 g (30.2 mmoles)sodium hydroxide in 50 ml absolute ethanol was degassed and refluxedunder N₂ until dissolution. The solvent was evaporated in vacuo andresidue dried at 0.05 mm Hg for 1 hour. The residue was dissolved in 50ml freshly distilled hexamethylphosphoramide (HMPA), 6.6 g (33.3 mmoles)of methyl α-bromo-β-methylbutyrate and 1 g sodium iodide added, themixture degassed and heated at 65° for 4 days under N₂. The cooledmixture was then poured into 400 ml of ice slurry and extracted with 3 ×100 ml ether. The combined ethereal extracts were washed wtih 100 ml 5%aqueous sodium carbonate, 100 ml water, 50 ml saturated aqueous sodiumchloride, dried over sodium carbonate, evaporated in vacuo and stored at0.05 mm Hg for one hour. The residue was dissolved in 400 ml dry etherand hydrogen chloride added until precipitation ceased. Filtration andwashing with 1 liter of dry ether yielded a white powder which wasdissolved in 100 ml water and made basic with sodium carbonate solution.The resulting oil was taken up in 2 × 200 ml ether, dried over magnesiumsulfate, evaporated in vacuo and placed on pump overnight to give 5.4 g(45%). A yellow oil cyrstallized on standing. m.p. 98°-107°.

B. The above prepared ester (450 mg, 1.2 mmoles) was refluxed in 10 ml 2N hydrochloric acid for 3 hours. The reaction mixture was evaporated invacuo, the vacuum maintained for 1 hour. The residue was taken up in 10ml water and the pH adjusted to 6 with 2 N sodium hydroxide. Theresulting suspension was centrifuged until clear and the supernatantdecanted and washed with 20 ml of ether, 20 ml of benzene, thenevaporated in vacuo and dried with a pump vacuum for 1 hour. Hot ethanol(abs.) (2 ml) was added to the residue and the resulting brownsuspension was centrifuged and the supernatant decanted into 10 ml ofacetone. Filtration and washing with 10 ml of a 2:1 mixture ofacetone:abs. ethanol yielded 100 mg (22%) of off-white crystals.

C. Into a reaction vessel was introduced 16.9 mg (44 μmole) of theα-isopropyl carboxymethylmorphine prepared above, 5.25 μl (40 μmoles) ofisobutyl chloroformate and 1 cc of dimethyl formamide and the mixtureallowed to stand for one hour at -15°.

D. A solution of 0.8 ml of 4 mg of porcine heart MDH in 0.1 M phosphate-- 0.05 M carbonate buffer, pH 9, was combined with 50 μl of the abovesolution and the pH maintained at 9 with dilute NaOH. The mixture wasstirred for one hour at 4° and diluted with sufficient 0.05M phosphatebuffer, pH 7.5 to give a total volume of 5cc. The solution was thendialyzed against that buffer to provide α-isopropylcarboxymethylmorphineconjugated malate dehydrogenase.

EXAMPLE 1.9

Glucose-6-phosphate dehydrogenase (G6PDH) from Leuconostoc mesenteroideswas used. A solution of 4 mg of G6PDH (3.12 × 10⁻⁸ mole) in 0.05phosphate -- 0.025 M carbonate buffer, pH 9.0 was treated while stirringat 4° C with 25 μl of a 50 μmole/ml DMF solution of theisobutylchloroformate mixed anhydride of O-3-carboxymethylmorphine (¹⁴ Cin N-methyl). After one hour the solution was dialyzed exhaustivelyagainst 0.05 M phosphate, pH 7.5. Scintillation counting of an aliquotrevealed 9.3 haptens/G6PDH incorporated.

The assay mixture was constituted as follows:

    ______________________________________                                        0.055 M Tris-HCl-0.033 M MgCl.sub.2, pH 7.8                                                             0.900 ml                                            0.10 M NAD                0.020 ml                                            0.066 M Glucose-6-phosphate                                                                             0.050 ml                                            Enzyme solution           Variable                                            ______________________________________                                    

The reagents are mixed and the increase in absorbance at 340 nm is readin a spectrophotometer.

Data:

1. A 1/50 dilution of the stock enzyme solution in assay buffer wasprepared. Ten μl gave a rate of 0.125 OD/min.

2. Addition of 3 μl of 3.7 × 10⁻⁵ M (binding site concentration) opiateantibody prior to addition of 10 μl of enzyme gave a rate of 0.059OD/min.

3. Addition of 30 μl of 1.67 × 10⁻³ M codeine prior to addition of thereagents mentioned in 2 gave a rate of 0.125 OD/min.

EXAMPLE 1.10

A. To a solution of 5 g of morphine monohydrate in 40 ml of dry dimethylformamide (DMF) and 300 ml of acetone, 25 g of finely powdered potassiumcarbonate and 5 ml of chloroacetonitrile was added. The solution wasallowed to reflux for 20 hours, cooled to room temperature and filtered.The filtrate was washed three times with acetone-DMF in a 20:1 mixtureand then evaporated to dryness in vacuo. Methylene chloride (500 ml) wasadded and the mixture heated to reflux, filtered while hot and thefiltrate evaporated to give a brown oil. Addition of 200 ml of ethylacetate led to crystallization by cooling overnight in the icebox. Totalyield, 4.4 g of cyanomethylmorphine. m.p. 186°-188°.

B. To a solution of 1.0 g (3.09 mmoles) of cyanomethylmorphine in 50 mlof dry methanol was added 7 ml of a 0.0435 M sodium methoxide solutionin methanol. The reaction was allowed to stir at room temperature for 48hours. After this time, 18 μl of glacial acetic acid was added, thereaction was stirred and then evaporated to dryness. The residue wasdissolved with ethylene chloride and filtered, to remove sodium acetate.After filtration, the organic phase was evaporated to give 1.2 g of alight yellow salt.

C. A solution of 18 mg (0.5 mmoles) of O³ -methoxyimidoylmethylmorphinein 0.5 ml of dry DMF was added dropwise to a cold (0°) solution of 60 mgof lysozyme in 6 ml of water. The aqueous solution was first adjusted topH 7.5 with 0.05 M sodium hydroxide. The solution was then stirred at 0°overnight. The pH was adjusted to 7 and the aqueous solution wasdialyzed against water for 48 hours. The resulting dialysate wassuitable for enzyme immunoassay. It could be inhibited by morphineantibodies, and full recovery of activity could be achieved upon theaddition of an aqueous solution of morphine.

EXAMPLE 1.11

To 4 mg of glucose 6-phosphate dehydrogenase (L. mesenteroides) in 0.05M sodium phosphate, pH 8.5, was added 75 μl of a solution containing 73mg of O³ -methoxyimidoylmethyl morphine per ml of DMF (200 μmole per ml)at 4°. The pH was maintained at 8.5 by addition of dilute HCl as needed.The reaction was allowed to proceed for 4 hours, after which thesolution is exhaustively dialyzed against 0.055 M Tris HCl-0.033 Mmagnesium chloride, pH 7.8. The solution was diluted to 8 cc with thesame buffer. The enzyme was found to contain 10.5 morphine groups permolecule.

The activity of 10 μl of a 1:50 dilution of this solution was found to0.099 OD/min. With addition of 5 μl of an opiate antibody, 1.3 × 10⁻⁴ Mbinding sites, the rate was 0.019 3OD/min. When 20 μl of 1.7 × 10⁻³ Mcodeine was added prior to adding the antibody, the rate was 0.102OD/min.

EXAMPLE 2.1

A. A solution of tetramethylene dibromide (32.4 g, 150 mmoles) in dryether (150 ml) was added to magnesium (10.9 g, 450 mmoles) in ether (80ml) at such a rate that the ether refluxed. The reaction was carried outunder argon. After the addition was completed, the reaction mixture wasboiled for one hour. A solution of2,2-diphenyl-4-dimethylaminovaleronitrile (I), (prepared according to J.W. CUSIC, J. Am. Chem. Soc., 71, 3546. (1949)) (8.4 g, 30 mmoles) in dryxylene (100 ml) was added during 30 min. at room temperature, and themixture was stirred at 55° C for 1 hour. The reaction mixture was cooledin an ice-water bath and CO₂ was passed through with fast stirring forfour hours. Water (200 ml) and concentrated HCl (100 ml) were added, themagnesium filtered off, and the filtrate was refluxed for 2 hours. Thecooled clear solution was washed with ether (3 × 150 ml) and extractedwith dichloromethane (3 × 150 ml). This extract was evaporated todryness, and the residue dissolved in 0.5 liter of 0.5 sodium hydroxide.

This solution was washed with ether (3 × 100 ml), made acidic with conc,HCl (150 ml), saturated with sodium chloride and extracted withdichloromethane (3 × 200 ml). Evaporation of the solvent left an oil(7.55 g, 60%) 6-keto-7,7-diphenyl-9-(dimethylamino)decanoic acidhydrochloride, which moves as a single spot on TLC (HCCl₃ :MeOH 8:2 and7:3).

    ______________________________________                                        U. V. Spectrum                                                                0.02% CF.sub.3 COOH                                                                               293 (Σ =540);                                                           264 (Σ =500);                                       λ.sub.max    259 (Σ =535);                                       ______________________________________                                    

B. The acid, 20.1 mg (50 μmoles) was dissolved in 1 ml drydimethylformamide, 2 drops of triethylamine were added, and the chilledsolution was treated with 6.5 μl of isobutylchloroformate as describedpreviously in other preparations.

C. Lysozyme (120 mg, 50 μmoles of lysine) was dissolved in 12 ml ofwater. The pH was adjusted to 10 with 0.05 N sodium hydroxide, andmaintained there during the dropwise addition of the mixed anhydridesolution. After 30 minutes additional stirring, the mixture wascentrifuged. The supernatant fraction remained homogeneous throughdialysis against water and contained the lysozyme conjugate to themethadone analog.

D. The assay employed is described in Example 1.3 for thecarboxymethylmorphine lysozyme conjugate. The compositions employed werean enzyme solution at a concentration of 1.6 × 10⁻⁵ M and an antibodysolution having a concentration of 3.66 × 10⁻⁵ M, based on bindingsites, and having a binding constant of 6.55 × 10⁷. The reagentsolutions were combined to have an enzyme concentration at 2 × 10⁻⁷, anantibody concentration based on binding sites of 2.3 × 10⁻⁷ and a totalvolume including 0.080 ml of urine of 0.800 ml. Readings were taken at40 seconds. Sensitivity to methadone was found to be 1 × 10⁻⁶ (0.35 μgper ml).

EXAMPLE 3.1

A. 4-Cyano-4-phenylpiperidine hydrochloride (2.23 g) was dissolved in 15ml water to which was added 4 ml of 50% aqueous potassium hydroxide. Theoil was extracted with 3 × 15 ml ether and the organic layers were driedover an anhydrous magnesium sulfate. Filtration and evaporation of thesolution gave a residue which was placed in a glass ampoule along with 3ml of methanol and 1.23 ml of 50% aqueous potassium hydroxide. Thesealed ampoule was heated to 165°-170° for 3.5 hours and diluted with 50ml water. After extraction with chloroform the aqueous phase wasneutralized with DOWEX 50-X8 (H⁺ form) to pH 6. Filtration andevaporation yielded a residue (0.82 g) which melted above 300°.Recrystallization from water and drying over phosphorous pentoxide gavea compound with m.p. 285°-286°.

B. 4-Carboxy-4-phenylpiperidine (1.8 g) was refluxed in 50 ml of 5%ethanolic hydrochloride for 4 hours. The residue on evaporation of thesolvent was dissolved in acetone and the insoluble part filtered off.From the acetone solution a viscous oil remained on evaporation whichcrystallized on standing; m.p. 107°-110° (1.068 g). It was used withoutfurther purification.

B'. A solution of about 7.2 g of 4-cyano-4-phenylpiperidinehydrochloride in 6 ml 66% sulfuric acid was heated to 45° and stirredfor 45 min. On cooling to 125° the solution became slightly moreviscous. The addition of alcohol (stem of the addition funnel below thesurface of the reaction mixture) lowered the temperature to 105°. It waskept there for 4 hours. During the first hour 20 ml alcohol were added,in the next hours 6 ml each. The alcohol vapors were removed by acontinuous distillation. At the end of the addition the temperature wasraised to 125° until condensate is no longer formed. The hot solutionwas poured into 6 ml water/40 g ice containing 8g sodium hydroxide.After extraction with 3 × 70 ml ether, drying over anhydrous magnesiumsulfate and removal of the solvent, an oil remained which was distilledat 112°-115°/0.2 mm Hg, 4.01 g (54%).

C. 4-Carbethoxy-4-phenylpiperidine (4.01 g) was dissolved in 13 mlabsolute alcohol and refluxed together with 2.01 g sodium chloroacetate.After 7 hours no starting material was present as evidenced by TLC. Theprecipitated sodium chloride was removed by filtration and washed with 3ml ethanol. On cooling of the filtrate white crystals appeared.Filtration and drying gave 2.9 g (58%) of the title compound. m.p.138°-140° C. Evaporation of the mother liquor gave a glass which did notcrystallize from acetone/hexane.

D. To a solution of 29.7 mg (0.1 mmole) of4-carbethoxy-1-carboxymethyl-4-phenylpiperidine in 1.0 ml dry dimethylformamide at 0° was added 13.1 μl isobutyl chloroformate (0.1 mmole).The mixture was stirred at 0° for one hour.

E. The cold solution of mixed anhydride (prepared above) was addeddropwise to a solution of 100 mg lysozyme (6.9 μmole) and 100 mg sodiumbicarbonate in 10 ml water at 0°. The reaction was stirred at 4° for 24hours then dialyzed against water for 48 hours. The water was changedthree times a day. The partially purified enzyme conjugate was thenchromatographed on Bio-Rex-70 using a 0.05-0.20M pH 7.15 phosphatebuffer gradient. Lysozyme activity in the eluent was followed by theconventional lysozme assay employing Micrococcus lysodeikticus (20 mg/50ml buffer). The addition of γ-globulin from rabbit or sheep immunizedwith the BSA-conjugate of the meperidine acid caused thelysozyme-meperidine conjugate to be nearly completely inhibited.Addition of free meperidine to the inhibited enzyme-antibody complex ledto restoration of lysozyme activity.

EXAMPLE 4.1

A. Amphetamine sulphate (3.68 g, 20 mmoles of amine) was dissolved in0.5 N sodium hydroxide (80 ml). The alkaline solution was extracted withether, the ether dried and evaporated. The residue was dissolved inbenzene (50 ml) and diisopropylethylamine (3 ml) was added followed byethylbromoacetate (2.2 ml, 20 mmoles). The reaction mixture was refluxedfor one hour, cooled, filtered and the filtrate evaporated. The residuewas taken up in ether, washed several times with water, the ether driedand evaporated. The pure amino-ester was obtained by columnchromatography on silica (hexane:ether 7:3). Yield 3.1 g (70%), NMR andIR agree with the structure.

B. The amino-ester (2.5 g, 11.3 mmoles) was dissolved in 1:1 mixture ofmethanol and 1N sodium hydroxide (50 ml) and left at room temperatureovernight. The mixture was evaporated to a small volume, washed twicewith ether (2X25 ml) and acidified to pH 6 with conc. HCl. The crystalsthat separated out were recrystallized from ether-acetone to give twofractions: 900 mg, m.p. - 222°-25° (m.p. lit. 220°-5° C, Tetra. Letters.1966, 4603-7) and 450 mg, m.p. 219°-218°. Only the first fraction wasused for further reactions. λ_(max) ^(H).sbsp.2^(O) 257 nm, ε=159.

C. Amphetamine-carboxylic acid (700 mg, 3.8 mmoles) was suspended in drydioxane at 50° (50 ml) and phosgene (12.5 wt.%) in benzene (20 ml) wasadded in one portion. The reaction mixture was stirred at 40°-50° C for31/2hours, evaporated to dryness and redissolved in dry dioxane (20 ml).This solution was kept on ice for the next step.

D. N-carboxymethyl amphetamine (25 mg, 0.133 mmole) was suspended in 1.8ml of dry dioxane at 40° C. Phosgene (12.5 volume percent in benzene)(0.715 ml) was added in one portion. The reaction mixture was stirred at40° for 31/2 hours before an additional 0.2 ml of phosgene (12.5 volumepercent in benzene) was added. After stirring an additional 30 minutesthe solution became homogeneous. The solvent was removed in vacuo at 25°in the hood. An additional 0.70 ml of dry dioxane was added for use inthe next step.

E. The cold dioxane solution of the above product was added dropwiseover 5 minutes to a stirred, cold (0°) solution of 200 mg sodiumbicarbonate and 100 mg lysozyme in 10 ml water. The milky reactionmixture was stirred at 4° for 48 hours and then dialyzed against water(1 liter changed three times daily) for 48 hours. The residue waslyophylized and the residue used for activity and inhibition studies.

EXAMPLE 5.1

A. Sodium phenobarbital (5.08 g, 0.02 moles), methyl chloracetate, (2.16g, 0.02 moles) methanol (14 ml) and a catalytic amount of DMF (1 ml)were refluxed for 2 hours. A white precipitate separated out during thisperiod. The reaction mixture was cooled to room temperature andfiltered. The methanolic filtrate was evaporated to dryness to yieldabout 5 g of a gummy material which solidified on standing. (Theprecipitate from the above filtration partially dissolved when rewashedwith distilled water. The water-insoluble material, about 50 mg, provedto be the dialkylated product).

The solidified material was stirred with 20 ml of 1 N NaOH solution for15 minutes and then filtered. This separated the alkali-insolublederivatives, the monoalkylated product and unreacted phenobarbital. Thealkaline filtrate was acidified with conc. HCl to a pH 2 and the whitegummy precipitate which formed was taken up in methylene chloride.

Drying (MgSO₄) and evaporation of the organic solvent yielded 4 g ofgummy material. This was dissolved in benzene and chromatographed over acolumn of silica gel (40 g). Elution was with chloroform and 100 mlfractions were collected. (The progress of the chromatography wasfollowed by TLC, since the dialkylated product has an R_(f) 0.9, themonoalkylated material R_(f) 0.6 and phenobarbital R_(f) 0.1 withchloroform/methanol 95:5).

Fractions 2-5 combined yielded on evaporation 1.6 g of a gum whichsolidified on standing. Trituration with petroleum ether and filtrationyielded 1.5 g of a white powder which was shwon by NMR to be therequired monoalkylated derivative, N-methoxycarbonylmethylphenobarbital.

Further elution with chloroform (500 ml) yielded 1.5 g of a white solidwhich was shown to be unreacted phenobarbital.

B. The monoester prepared above (1 g) was refluxed with 10 ml of 20% HClsolution for 3.5 hours. The cooled reaction mixture was diluted withwater (20 ml) and extracted with ether. Evaporation of the other extractyielded 0.98 g of a colorless gum which very slowly solidifed onstanding. NMR and TLC showed that complete hydrolysis had occurred tothe acid.

A pure sample of the acid was prepared by preparative TLC for UVanalysis, with chloroform/methanol (5:1) as eluent. C. To a cold (0°)solution of 29.6 mg N-carboxymethyl phenobarbital (0.1 mmoles) and 14.3μl triethyl amine (0.1 mmoles) in 1.0 ml dry dimethyl formamide wasadded 13.1 μl isobutyl chloroformate (0.1 mmoles). The solution wasstirred at 4° for 1 hour before use.

D. The cold solution of mixed anhydride was added dropwise with stirringto a cold (4°) solution of 0.100 g lysozyme 6.9 mmole) and 0.100 gsodium bicarbonate in 10 ml water. The resulting heterogeneous solutionwas stored at 4° for 48 hours before being dialyzed against water for 48hours. (The water was changed three times daily). The dialysate was thenchromatographed on Bio-Rex 70 employing a 0.05-0.20 M pH 7.15 phosphatebuffer gradient for elution.

E. The assay employing the phenobarbital conjugate had an enzymeconcentration in the enzyme conjugate stock solution of 1.71 × 10⁻⁵ M,an antibody concentration based on binding sites in the stock solutionof 1.66 × 10⁻⁵ and a binding constant for the antibody of 5.94 × 10⁷.The assay solution had a total volume of 0.800 ml, employed a urinevolume of 0.080 ml, had an enzyme concentration of 2.14 × 10⁻⁷ and anantibody concentration based on binding sites of 2.08 × 10⁻⁷. The assaywas carried out for 40 seconds and the sensitivity was found to be 0.3μg/ml, the minimum detectable amount.

EXAMPLE 5.2

A. Ozone was passed through a cooled (dry ice/acetone) solution ofsodium secobarbital (2.6 g, 0.01 mole) in methanol (250 ml). Afterozonlysis was completed (positive KI test), nitrogen was passed throughthe reaction mixture to remove all traces of ozone and then dimethylsulfide (7 ml) was added to the cold solution with a syringe and allowedto stand overnight at room temperature. After evaporation of thesolvent, the residue was diluted with water (20 ml), acidified withconc. HCl and extracted with chloroform (3 × 20 ml). The chloroformextract was dried (MgSO₄) and evaporated to yield 2.4 g of gummycolorless material. NMR showed the presence of an aldehyde group at 89.7ppm. This was used without further purification in the reaction withmalonic acid.

B. A sample of pure aldehyde (0.24 g, 1 mmole), malonic acid (0.21 g, 2mmoles) 20 ml pyridine and 1 ml piperidine were refluxed together for 6hours. The solvent was removed on the flash evaporator and the residuedissolved in 10% sodium bicarbonate solution. The bicarbonate solutionwas washed with ether (3 × 20 ml) and then acidified with conc. HCl.Extraction with ether (2 × 20 ml) and then with chloroform (2 × 25 ml)followed by drying (MgSO₄) and evaporation of the combined organiclayers yielded 0.23 g (80% yield) of a white solid shown by NMR to bethe desired acid 5-(γ-crotonic acid)-5-(1'-methylbutyl) barbituric acid.Recrystallization from CHCl₃ /CCl₄ yielded 0.16 g of pure material.

C. To a solution of 5-(α-crotonic acid)-5-(1'-methylbutyl) barbituricacid, (0.282 g, 1 mmole) in DMF (3 ml), cooled to -15° (ice-salt bath)there was added triethylamine (0.28 ml, 2 mmoles) andisobutylchloroformate (0.13 ml, 1 mmole). Stirring was continued at -15°for 15 minutes and then at 0° for 30 minutes. The reaction mixture wasthen added dropwise, with a syringe, to a cooled solution of BSA (400mg) in water (56 ml) containing NaHCO₃ (2.6 g). The reaction mixture wasstirred at 0° (cold room) for 5 days when initial trubidity had nearlyall disappeared. The solution was then dialyzed against 4 1. ofphosphate buffer (pH 8) followed by distilled water to yield the desiredconjugate.

D. Lysozyme, 240 mg (100 μmoles of lysine) was dissolved in 20 ml ofwater and the solution chilled to 0° C. The solution was adjusted to pH10.2 with 0.05 N sodium hydroxide and the mixed anhydride (100 μmoles)in 1.5 ml dry dimethyl formamide added dropwise while the solution waskept between pH 9.6 - 9.9 by addition of base as required. The pH wasmaintained at 9.6 for another 30 minutes, after which time the mixturewas centrifuged.

The supernatant was dialyzed against 0.05 mole Tris-maleate, pH 8.0. Thepellet formed by centrifugation dissolved in 20 ml 8M urea, and wasdialzyed as described above, yielding additional amounts of enzyme. Theurea dialysis treatment was repeated until only 10 mg of insolublematerial remained.

E. An enzyme stock solution was prepared of the secobarbital conjugateto lysozyme having a concentration of enzyme of 2.08 × 10⁻⁵ M. Theantibody stock solution was 1.42 × 10⁻⁵ M based on binding sites, andthe anitbody had a binding constant of 8.4 × 10⁷ by FRAT^(R). In theassay solution, the enzyme concentration was 1.56 × 10⁻⁷ M, the antibodyconcentration based on binding sites was 2.66 × 10⁻⁷ M, the total assayvolume was 0.800 ml, the urine volume 0.080 ml, the time for the assay40 seconds, and the sensitivity found to 0.2 μg/ml.

EXAMPLE 5.3

Sodium hydride (0.85 g of a 50% oil paste, 18 mmoles) was added in smallamounts to a stirred solution of glutethimide, (3.7 g, 17 mmoles) in dryDMF (10 ml). Stirring was continued for about 5 minutes, when gasevolution was no longer observed. Sodium chloroacetate (2.2 g) was thenadded and the reaction mixture was stirred with heating in an oil bathat 100° for 3 hours. After cooling, the reaction mixture was dilutedwith 50 ml water, acidified with conc. HCl and then poured into 200 mlether. The ether layer was spearated and washed with water (2 × 50 ml).The organic layer was dried (MgSO₄) and evaporated to yield 3.4 g of awhite solid. Recrystallization from carbon tetrachloride/methylenechloride yielded the analytical sample of the acid.

Anal. Calcd. for C₁₅ H₁₇ NO₄ : C, 65.44; H, 6.22; N, 5.08 Found: C,64.92; H, 6.20; N, 4.99.

The N-carboxymethyl glutethimide can be conjugated to lysozyme as setforth in the conjugation for the barbitals. Antibodies can be preparedby conjugating the N-carboxymethyl glutethimide to bovine serum albumin(BSA) and injecting the conjugated BSA into animals to obtain theappropriate antibodies. The assay is carried out in the same manner aspreviously described for lysozyme.

EXAMPLE 5.4

A. To a suspended solution of sodium phenobarbital (1.0 g, 3,93 mmoles)in dry dimethylformamide (12 ml) was added ethyl-5-bromovalerate (920mg, 4.43 mmoles) with stirring, and the mixture was heated at 40° for 10minutes to give a clear solution. The mixture was stirred at roomtemperature for 15 hours, and then potassium iodide (200 mg) was addedto complete the reaction, which was followed by TLC (silica gel, 5%methanol -- 95% chloroform). Most of the solvent was evaporated underreduced pressure to leave an oil, which was dissolved in dichloromethane(50 ml) and washed with water. The solution was shaken once with 2.5wt.% sodium carbonate solution (25 ml) to remove unchanged startingphenobarbituric acid. The dichloromethane layer, after being washed withwater and dried over anhydrous sodium sulfate, was evaporated to leavean oil (1.4 g). This oil was separated into two fractions by preparativeTLC, silica gel. The oil was developed with 5% methanol - 95%chloroform, and each fraction was collected by cutting and extractedwith acetone. The products, after removal of the solvent, were dissolvedin dichloromethane, washed with water, and dried over anhydrous sodiumsulfate. One fraction (R_(f) 0.7) gave a colorless oil (0.5 g, 36%)which proved to be analytically pure monoalkylated compound II by IR andPMR spectra, and microanalysis:

Anal. Calcd. for C₁₉ H₂₄ O₅ N₂ : C, 63.32; H, 6.71; N, 7.77; Found; C,63.35; H, 6.75; N, 7.86.

B. The ethyl ester prepared above (120 mg, 0.333 mmole) was dissolvedinto a mixture of conc. hydrochloric acid (2.5 ml) tetrahydrofuran (5ml) and water (1 ml), and then kept at room temperature overnight. Afterevaporation of tetrahydrofuran under reduced pressure, the residue wasdiluted with saturated sodium chloride solution (10 ml) and extractedwith dichloromethane. The dichloromethane layer was extracted withsaturated sodium bicarbonate solution, and the combined alkaline layers,after being carefully acidified with conc. hydrochloric acid in an icebath, were extracted with dichloromethane. The dichloromethane solutionwas washed with water, dried over anhydrous sodium sulfate, andevaporated to dryness to give an oily residue (100 mg, 93%), whichcrystallized on standing. Recrystalliztion from ether/n-hexane gave ananalytical sample of the desired acid.

Anal. Calcd. for C₁₇ H₂ O O₅ N₂ : C, 61.43; H, 6.07; N, 8.43; Found: C,61.48; H, 6.08; N, 8.43.

C. A sample of the above prepared acid was dried overnight under vacuumat 80° C before use.

In a flask protected from moisture, 166 mg of the acid was dissolved in5 cc of dry DMF and 150 μl of triethylamine added. The solution wascooled to -15° and then 65 μl (0.5 mmole) of isobutyl chloroformateadded. The mixture was stirred for one hour with the temperaturemaintained between -5° and 0°.

Lysozyme (1.2 g, 0.5 mmole lysine) was dissolved in 80 ml distilledwater in a beaker equipped with a magnetic stirrer. The solution wascooled in an ice-water bath to 4° and the pH was adjusted to 9.5 with0.5 M NaOH. The anhydride reaction mixture prepared above was addeddropwise with stirring. The pH was kept at 9.5 - 9.7 during thisaddition by the slow addition of 0.5 M NaOH. The solution was stirred anadditional 90 minutes at 4°.

The pH was then lowered to 8.5 by the addition of 1 M HCl and themixture centrifuged at 12,000 rpm for 20 minutes. The supernatantfraction (S) was dialyzed against 6 changes of 0.05 M Tris buffer pH8.0. The precipitate on being stirred briefly with 100 ml 8 M ureadissolved completely (P₁). Upon dialysis (as for S) a significant amountof material came out of solution. The precipitated material, separatedby centrifugation, was redissolved in urea and redialyzed (P₂, P₃, P₄fractions). The various fractions of soluble enzyme (P₁, P₂, P₃, P₄,etc.) were all tested for inhibition with phenobarbital antisera (equalamount of antisera was used with all fractions). The fractions P₁, P₂,P₃ and P₄ showed inhibitions of 77, 85, 86 and 92% respectively. A poolof P₂, P₃ and P₄ was prepared for use in the assay.

EXAMPLE 5.5

A sample of the secobarbital acid prepared as described in Example 5.2was dried overnight under vacuum at 80° before use.

In a flask equipped with a magnetic stirrer and a drying tube wasdissolved 140 mg of the above acid in 5 ml dry DMF. After the additionof 137 μl dry triethylamine the mixture was cooled to -15° and 68.5 μlof isobutyl chloroformate added. The reaction was stirred at -5° to 0°for 1 hour and then conjugated to lysozyme.

A solution of lysozyme (1.2 g, 0.5 mmole lysine) in 80 ml distilledwater was cooled in an ice bath to 4° and the pH adjusted to 9.5 with0.5 M NaOH. The acid anhydride prepared above was added dropwise withstirring as the pH was maintained at 9.5 - 9.7 by the slow addition of0.5 M NaOH. The heterogeneous reaction mixture was allowed to stir anadditional 90 minutes at 4° before the pH was lowered to 8.5 with 1 MHCl. The mixture was centrifuged at 12,000 rpm for 20 minutes. Theprecipitate, on being stirred with 100 ml 8 M urea dissolved completelybut a significant amount of material came out of solution duringdialysis (6 changes with 0.05 M tris at pH 8.0). The dialysate wascentrifuged and the supernatant (P₁) retained. The pellet was againsuspended in 8 M urea and dialyzed, and in this manner a number ofsoluble enzyme fractions (P₂, P₃. . . P_(n)) were obtained. Theprecipitate fractions were all tested for their ability to be inhibitedwith seconal antisera (the same amount of antisera was used forfractions). Inhibition of activity of 75, 80, 91 and 93% was obtainedfor fractions P₁, P₂, P₃ and P₄ respectively. The P₁ fraction was notconsidered suitable for use in the assays, but fractions P₂, P₃ and P₄were all combined and used as a pool.

EXAMPLE 6.1

A. Cocaine (5 g) was refluxed in 25 ml water for 6.5 hours. Theremaining oil after evaporation of the solution was dissolved in 5 mlhot water. On cooling long, white crystals separated (2.87 g). Another543 g were obtained from the mother liquor.

B. Benzoylecgonine (1 g) was refluxed in 25 ml 2N hydrochloric acid for1 hours. After cooling, the solution was filtered and extracted withether. The aqueous phase was neturalized with sodium bicarbonate to pH5.8. On evaporation a white residue remained which was refluxed with 40ml ethanol (95%), filtered and the solvent evaporated. The oily residue(580 mg) recrystallized on addition of 0.5 ml ethanol (130 mg). m.p.195°-197° (decomp).

C. To a suspension of 22.7 mg (0.1 mmole) ecgonine hydrochloride in 1.0ml dry dimethyl formamide at 0° was added 13.7 μl isobutyl chlorformate(0.1 mmole). The mixture was stirred at 0° for 2 hours and then used forthe conjugation.

D. To a cold (4°) solution of 100 mg lysozyme (6.9 μmole) and 100 mgsodium bicarbonate in 10 ml water was added the dimethyl formamidesuspension of the mixed anhydride. The homogeneous solution was stirredat 4° for 40 hours and then dialyzed against water (1 liter changedthree times daily) for 4 days. The dialyzed material was thenlyophylized to dryness. Activity and inhibition studies were performedon this material.

EXAMPLE 6.2

p-Aminobenzoyl ecgonine (50 mg.) in 1 ml. of 0.2 N HCl at 0° was addeddropwise to 11.3 mg. NaNO₂ in 1 ml. H₂ O at 0°. A yellow colordeveloped. The resulting diazonium salt was added dropwise over 5 min.to a solution of 200 mg. lysozyme (Miles 6 × recryst.) in 10 ml. waterat 0°, pH 9.0. A red color developed, and some precipitate appeared. ThepH was maintained at 9.0 with stirring, 1.5 hrs. at 0°. The mixture wasthen centrifuged.

The supernatant was yellow, and the precipitate red. The precipitate wasreadily dissolved in 8 M urea. Both fractions were dialyzed against H₂O.

The supernatant fraction was recovered from the dialysis and tested forinhibition and sensitivity towards benzoyl ecgonine.

Assay

1. Antibody omitted (50 μl) Rate is 168; 171 OD/min.

2. Antibody included (1:1) Rate is 45 OD/min.

3. (2) + 0.5 μg/ml. benzoyl ecgonine Rate is 50;52 OD/min.

4. (2) + 5.0 μg/ml benzoyl ecgonine Rate is 70;75 OD/min.

5. (2) + 50 μg/ml benzoyl ecgonine Rate is 122;125 OD/min.

EXAMPLE 7.1

A. To a suspension of 4.1 g (30 mmoles) of p-aminobenzoic acid in 300 ccof water was added 4.5 cc of concentrated hydrochloric acid. Thesuspension was warmed slightly to hasten solution, at which time anaddition of 9 cc of concentrated hydrochloric acid was added and theslution cooled to 0° - 5°. To this stirring solution was added at once aprecooled solution of 2.1 g of sodium nitrite in 6 cc of water. After 15minutes at 0° a test with starch iodide paper indicated excess nitrousacid. The pH was raised to the pH range of congo red by the addition ofsaturated aqueous sodium acetate. To this solution was added at once 6.3g of sodium sulfite in 15 cc of water. A certain amount of color wasproduced at this step and some precipitation was observed as well. Thesolution could be assayed for active diazonium salt or the reactive synisomer, (1) by touching a drop to filter paper which had been treatedwith β-naphthol in aqueous alcoholic carbonate solution. Aninstantaneous red color signaled the presence of the reactive species.After one hour at room temperature, this spot test showed no activediazonium species remaining. The aqueous solution was treated withdecolorizing charcoal and filtered. The addition of solid sodiumchloride to saturation caused the precipitation of the desiredanti-diazosulfonate as a yellow crystalline solid which is shown byspectroscopic techniques to be the monohydrate.

B. To 500 mg of disodio para-(anti-diazosulfonato) benzoate (1.7 × 10⁻³moles) and 10 ml of dry dimethyl formamide (DMF) cooled in an ice bathto 0° was added one ml of isobutyl chloroformate (7.61 × 10⁻³ moles)followed by 1.5 ml of triethylamine. The mixture was stirred for 4 hoursat 0° followed by standing for 2 days in a cold room with stirring.Excess chloroformate and dimethylformamide were removed by rotaryevaporation at 40°.

C. The supernatant resulting from slurrying 30 mg of insulin with pH 8.8tris-barbital buffer was combined with 50 λ (6.85 × 10⁻³ mmoles) of theanhydride prepared above. The mixture was stirred for 2.5 hours at 4°,at the end of which time, the solution was dialyzed against pH 8.8tris-barbital buffer.

The above solution was then combined with 0.25 ml (10 mg/ml, 3.4 × 10⁻⁵mmoles) of dialyzed malate dehydrogenase (dialyzed against tris-barbitalbuffer, pH 8.8) and the solution irradiated with visible light (greaterthan 398 nm) for approximately 45 minutes. A small sample was combinedwith β-naphthol, the solution turning red, indicating that all thediazosulfonate had not reacted.

The resulting product was chromatographed through a column of SephadexG-50 swelled with bicarbonate pH 8.8 buffer. Five λ of the solution hadan activity when assayed for malate dehydrogenase of about .12 OD/min.

EXAMPLE 8.1

The testosterone-3-carboxymethyloxime, 36.1 mg (100 μmole), wasdissolved in 1 ml of dimethylformamide containing 3 drops oftriethylamine. The solution was cooled to -15° C and 13.1 μl (100 μmole)of isobutylchloroformate were added. Stirred for 1 hour at -15° to -5° Cduring which time the solution turned light orange.

Malate dehydrogenase, 0.5 cc of 10 mg/ml suspension in 2.8M ammoniumsulfate (5 mg MDH, 6.8 × 10⁻⁸ mole MDH, 4.4 × 10⁻⁶ mole lysine residues)was centrifuged at 15,000 rpm for 20 min. The pellet was dissolved in 1ml of water and the solution was dialyzed against water at 4° C for 5hours (3 changes). The solution was brought to pH 8.5 with dilute NaOHat 4° C and 44 μl of the mixed anhydride solution (4.4 mmole mixedanhydride; corresponds to 1 hapten per lysine) was added to the stirredenzyme solution in three portions over 5 minutes. Sodium hydroxidesolution was added as needed to keep the pH at 8.5. Initially thesolution was turbid, but cleared during 1 hour stirring at 4° C.

The solution was exhaustively dialyzed against 0.05M phosphate buffer,pH 7.5. A small amount of sediment was removed by centrifugation.

Assay: Because of the instability of highly diluted enzyme solutions,the stock solution (5 mg/ml; 3.4 × 10⁻⁵ M) was diluted 1 to 500 justprior to each assay. The order of addition of reagents to the assaymixture was as follows: 1)antibody (when used), 2)diluted enzyme,3)oxalacetic acid, 4)NADH. The final enzyme concentration was 2.7 × 10⁻⁹M. The antibody concentration was not known. Sufficient antibody wasused to achieve greater than 40% inhibition of the enzyme activity. Thiscorresponded to an equivalent of 10 μl of antibody containing serum.

(1) Enzyme Alone -- 0.073 OD/min. (2) Enzyme + Antibody -- 0.042 OD/min.(3) Enzyme + Antibody + 50 μl 10⁻⁵ M testosterone (added first) -- 0.073OD/min.

EXAMPLE 8.2

To 33.0 mg (10⁻⁴ mole) of 3-(O-carboxymethyl)estradiol dissolved in 1 mlof anhydrous dimethylformamide was added 2 drops of triethylamine. Thesolution was cooled to -15°, and 13.1 μ l (10⁻⁴ mole) ofisobutylchloroformate was added. The solution was maintained at -15° for1 hour.

The above solution (44 μl) was added to a solution of 5 mg malicdehydrogenase in 0.004 M Na₂ HPO₄, pH 9 which had been cooled to 4°.During the reaction the pH was maintained at 8.5 to 9.0 by adding sodiumhydroxide solution. The solution, turbid initially, cleared after 2hours. It was dialyzed exhaustively against 0.05 M sodium phosphate, pH7.5; then clarified by centrifugation.

Assay

The stock enzyme solution was diluted 1 to 1000 with 1M Na₂ HPO₄solution and assayed in the customary manner entailing following theoxidation of reduced nicotinamide adenine dinucleotide (NADH) in thepresence of oxaloacetic acid at 340 nm, 30° C. Anti-estradiol antibodieswere prepared in rabbits and the γ-globulin (4×10⁻⁷ M binding sites)fraction was used in this assay.

1. 20 μl of the enzyme solution has an activity of 0.107 OD/min.

2. Addition of 5 μl of the antibody solution reduced the activity to0.070 OD/min.

3. Addition of a) 5 μl of antibody, and b) 20 μl of the enzyme to theassay mixture containing 20 μl of 10⁻³ M estradiol gave 0.106 OD/min.

Assays

To further demonstrate the utility of the subject invention and itsversatility in being able to distinguish a wide range of differentcompounds and to quantitatively or semi-quantitatively determine theconcentration of these compounds in different physiological fluids, anumber of assays were carried out. In these assays, the sensitivity ofthe assays was determined as to minimum concentrations required fordetectable levels. Also comparisons were made with a wide variety ofcompounds to determine whether the antibodies employed would respond tocompounds other than those which were intended to be assayed. In manyinstances the results were checked not only by thin layerchromatography, but also by an electron spin resonance techniqueentitled FRAT^(R), supplied by Syva Corp.

As previously indicated, various protocols can be employed While theorder of addition is not crucial, one order is preferred, particularlywhere the binding of the receptor to the enzyme-bound-ligand is strongerthan the binding of the receptor to the ligand.

The preferred order is to combine the unknown medium with the receptor.The binding of ligand with receptor is rapid, so that the addition ofthe enzyme-bound-ligand may be made promptly after combining the unknownmedium and receptor, usually within a minute. After the addition of theenzyme-bound-ligand a short time interval is usually allowed to pass,and the enzymatic activity determined as the average rate over one-halfto a few minutes, usually fewer than five minutes.

In some situations, e.g., low ligand concentration, it may be desirableto measure enzymatic activity at equilibrium. For measurements atequilibrium, the binding constants of the ligand and enzyme-bound-ligandshould be within about one order of magnitude.

The first system to be considered is the morphine conjugate to lysozyme.Following the assay for lysozyme as previously described,cross-reactivities were carried out in order to determine whichcompounds other than morphine, the antibody would recognize, and wouldtherefore provide a positive result for the assay. The following tableillustrates the cross-reactivity for a number of compounds. The resultsshow, that the antibody will recognize those compounds which havesubstantially the same ring structure as morphine, but would notrecognize those compounds which do not have the same ring structure.

    ______________________________________                                        MORPHINE CROSS-REACTIVITY                                                                 Concentration                                                     Compound    μg/ml      --       % max rate                                 ______________________________________                                        Codeine     3           1 × 10.sup.-5                                                                      90                                                     0.3         1 × 10.sup.-6                                                                      7                                          Morphine    10          2.1 × 10.sup.-5                                                                    77                                         glucuronide 0.3         6.35 × 10.sup.-7                                                                   4                                          Morphine    3           1 × 10.sup.-5                                                                      67                                                     0.3         1 × 10.sup.-6                                                                      5                                          Hydromor-   3           1 × 10.sup.-5                                                                      55                                         phine       0.3         1 × 10.sup.-6                                                                      6                                          Thorazine   35          9.9 × 10.sup.-5                                                                    6                                          Methadone   30          8.7 × 10.sup.-5                                                                    1                                          Darvon      300         8 × 10.sup.-4                                                                      4                                          Cocaine     300         1 × 10.sup.-3                                                                      2                                          Pentazo-                                                                      cine        300         7.5 × 10.sup.-4                                                                    3                                          Phenobar-                                                                     bital       300         1.2 × 10.sup.-3                                                                    0                                          ______________________________________                                    

A total of 91 samples of patient urine were taken from a methadoneclinic. All of the urines had been checked by thin layer chromatography,without hydrolysis, and four were found to contain morphine. Wheremorphine is present as the glucuronide it is not detected by thechromatographic system normally employed. The urines were then tested,both by FRAT® and the subject enzyme assay. Of the 91 samples, both theFRAT® and the subject enzyme assay showed the same 17 samples to bepositive, which included the four positive samples found by thin layerchromatography. The amounts of morphine detected varied from a low ofabout 0.3 μg/ml to a high of about 14.3 μg/ml.

Morphine has also been assayed in saliva. The following reagentsolutions were prepared: 0.1 M potassium phosphate buffer solution, pH7.5, containing 100 mg per liter of disodioethylene diamine tetraaceticacid; 14 mM nicotinamide adenine dinucleotide (NADH); 7 mM oxaloacetatein potassium phosphate buffer; morphine antibody having a bindingconstant as determined by an electron spin resonance technique (FRAT®,supplied by Syva Corp.) of 2.1 × 10⁷ and at a concentration based onbinding sites of 3.4 × 10⁻⁵ M and malate dehydrogenase bound O³-α-isopropylcarboxymethylmorphine in 1 M potassium phosphate buffercontaining 0.25% bovine serum albumin as a stabilizer.

The assays were carried out at 37° C and 340 nm. The level of inhibitionvaried with time, so that the assay is carried out for one minute of aspecific period, usually after two minutes of mixing the reagents.

The saliva which is employed in the assay is prepared as follows, inorder to destroy any malate dehydrogenase activity present in the salivaand reduce the viscosity of the saliva, 0.05 ml of saliva is combinedwith 0.5 ml of 0.036 N HCl, mixed, and the mixture allowed to stand twominutes at ambient temperature. To the mixture is then added 0.1 ml of0.018 N NaOH, the mixture agitated and then centrifuged for 10 minutesat 12,000 G at 0°. The saliva is then ready for use in the assay.

Into a 2 ml cup is introduced 350 μl of the 0.1 M potassium phosphatebuffer, 20 μl of NADH, 150 μl of the oxaloacetate, 200 μl of the treatedsaliva, 10 μl of the antibody solution and 20 μl of the malatedehydrogenase bound O³ -α- isopropylcarboxymethylmorphine, with themeasuring sampler being rinsed with 250μl of the 0.1 M potassiumphosphate buffer after addition of antibody and enzyme to insureaccurate transfer.

The rate in the period from the second minute to the third minute isthen determined from the zero time of the introduction of the mixtureinto the spectrophotometer. The rate is determined by subtracting thechange in optical density (OD) of the enzyme inhibited with antibodyfrom the OD of the sample and this value divided by the value obtainedby subtracting the OD of the inhibited enzyme from the OD of theuninhibited enzyme. The value for the uninhibited enzyme is an averageof the first and second minute reading, where the second minute readingis employed for the rate determination of the sample.

It was found appropriate to clean the cuvette employed in thespectrophotometer with dithioerythritol after each use.

Twenty-one samples were spiked with morphine. Of the samples spiked,with 5 × 10⁻⁷ M morphine, the results ranged from 2.8 to 5.0 × 10⁻⁷,only 5 of the results being below 4 × 10⁻⁷ M. When the samples werespiked with 5 × 10⁻⁸ M, the results varied from 4.0 to 10 × 10⁻⁸, withmost of the results being between 4 and 7.5 × 10⁻⁸.

The next ligand to be considered is methadone. The methadone lysozymeconjugate was employed in the normal assay for lysozyme. The followingtable indicates the results from the cross-reactivity study. The resultsare reported as relative reactivity to methadone at 0.5 μg/ml. That is,the relative activity is the ratio of the concentration of the drug inquestion to the concentration of methadone necessary to give the sameoptical density reading. The smaller the relative reactivity, the lessreactivity the compound has in the assay. While a large number ofcompounds were studied, only a few compounds showed any response. Thefollowing table indicates the results.

    ______________________________________                                        METHADONE CROSS-REACTIVITY                                                    Compound         Relative Reactivity                                          ______________________________________                                        Methadone        1                                                            Chlorpromazine   0.011                                                        Dextromethorphan 0.0042                                                       Dextropropoxyphone                                                                             0.00089                                                      Phenergan        0.0125                                                       ______________________________________                                    

A group of 12 urines was collected from known heroin addicts. The urineswere all shown to be positive morphine and negative methadone by thinlayer chromatography. However, when the urines were assayed by the FRAT®method as well as by the subject enzyme method, both of theseimmunoassay techniques agreed on the presence of methadone in three ofthe samples, as well as the positive presence of morphine in all thesamples. When 100 samples were taken from a methadone clinic, there was100% agreement between the FRAT® method and the enzyme immunoassaymethod as far as positive or negative for the presence of methadone andsubstantially good quantitative agreement between the determinations bythe two different methods.

The next drug to be considered is amphetamine. Again, the same procedureis employed for lysozyme as for the prior assays, except that 50 μl ofurine is employed. The relative reactivity is related to 1 μg/mlamphetamine.

    ______________________________________                                        AMPHETAMINE CROSS-REACTIVITY                                                  Compoound        Relative Reactivity                                          ______________________________________                                        Amphetamine      1                                                            Methamphetamine  1.1                                                          Mephentermine, phentermine                                                                     0.62                                                         Propylhexedrine  0.05                                                         Phenethylamine   0.2                                                          Cyclopentamine   0.33                                                         Ephedrine        0.22                                                         Phenylpropanolamine                                                                            0.33                                                         Nylidrin         0.27                                                         Isoxsuprine      0.2                                                          p-Hydroxyamphetamine                                                                           0.022                                                        ______________________________________                                    

A series of urine samples were obtained which were verified to containamphetamine by gas-liquid chromatography. The samples were then assayedby both the FRAT® assay and the subject enzyme assay. Excellentquantitative agreement was obtained between the FRAT® assay and thesubject amphetamine assay.

It is found that phenylpropanolamine which has a relatively highrelative reactivity is available in over-the-counter prescriptions. Inorder to avoid false positives, when a positive result is obtained froma urine, the urine is treated with sodium periodate and tetramethylammonium hydroxide for a few minutes at ambient temperatures. The pH ofthe urine should be in the range of about 8 to 9. This treatment iseffective in removing phenylpropanolamine as an interferant. The othercompounds which cross-react are not of a sufficient occurrence to be ofsubstantial concern.

The next system is the barbiturate system. The cross-reactivity forphenobarbital and secobarbital assays are provided in the followingtable for the reagents of Examples 5.1 and 5.2.

    __________________________________________________________________________    BARBITAL CROSS-REACTIVITY                                                             Concentration                                                                            Phenobarbital                                                                         Concentration                                                                             Secobarbital                           Compound                                                                             μg/ml                                                                            M × 10.sup.5                                                                  % Max Rate                                                                            μg/ml                                                                            M × 10.sup.5                                                                  % Max Rate                             __________________________________________________________________________    Phenobarbi-                                                                          2.54  1     42.4    2.54  1     0                                      tal    0.254 0.1   10.0    0.25  0.1   0                                      Secobarbi-                                                                           13    5     16.7    2.6   1     30.1                                   tal    1.3   .5    9.9     0.26  0.1   3.3                                    Amobarbi-                                                                            2.5   1     16.7    2.48  1     16.3                                   tal    0.25  .1    6.1     0.25  0.1   3.3                                    Tabutal                                                                              3     1.34  12.4    3     1.34  9.1                                           0.3   .13   3.9     0.3   0.13  1.4                                    Thiopental                                                                           3     1.13  15.8    3     1.13  16.0                                          0.3   .11   4.8     0.3   0.11  1.4                                    Glutethi-                                                                            21.7  10    9.0     21.7  10    0.8                                    mide   2.17  1     1.4                                                        Morphine                                                                             285   100   2.2     285   100   2.8                                    Demerol                                                                              284   100   0       284   100   2.1                                    Diphenyl                                                                      hydantoin                                                                            300   100   18.4    300   100   7.7                                    __________________________________________________________________________

Comparison of results of the subject enzyme assay with results obtainedfrom thin layer chromatography were in agreement with one exception asto the presence or absence of barbiturates. By using the two differentantibodies for the two barbiturates, a qualitative judgment could bemade of the class of barbiturates present.

A barbiturate assay was carried out by combining antibodies tophenobarbital and secobarbital and the lysozyme conjugate prepared asdescribed in Examples 5.4 and 5.5. The phenobarbital-lysozyme conjugate(11 μl, 1.066 × 10⁻⁵ M) was combined with 4.65 μl (1.89 × 10⁻⁵ M) of thesecobarbital-lysozyme conjugate, 5 μl of 1% BSA in pH 6.0 tris-maleate0.025 M buffer and 29.35 μl of pH 5.0 tris-maleate 0.025 M buffer toprovide 50 μl of reagent with a maximum rate of ≈ 300 OD/min. (enzymeunits). The antibody solution was prepared by combining 22.8 μl ofphenobarbital antibody (1.03 × 10⁻⁵ M based on binding sites) with 15.4μl of secobarbital antibody (1.14 × 10⁻⁵ M based on binding sites) and11.8 μl of pH 7.4 tris- maleate 0.025 M buffer.

The assay for lysozyme was carried out in the conventional manner,employing 50 μl of urine.

A group of 21 barbiturate positive urine samples were collected andanalyzed by thin layer chromatography (TLC), gas-liquid chromatography(GLC), FRAT® and the subject enzyme assay technique. The following tableindicates the results.

                                      TABLE                                       __________________________________________________________________________                                    Enzyme                                            Independent    GLC    PRAT ®                                                                          Assay                                         Sample                                                                            Laboratory                                                                            TLC    μg/ml                                                                             μg/ml                                                                            μg/ml                                      __________________________________________________________________________    1   pentobarbital                                                                         pento- pento- 15    5.8                                               unidentified                                                                          barbital                                                                             barbital                                                       barb           (3.5)                                                          methadone                                                                 2   phenobarbi-                                                                           phenobar-                                                                            phenobarbi-                                                                          1.6   2.15                                              tal     bital  tal (1.0)                                                      methadone      butabarbital                                                                  (2.0)                                                      3*  phenobarbi-                                                                           phenobar-                                                                            phenobarbi-                                                    tal     bital  tal (15.0)                                                                           9.0   4.7                                               methadone                                                                 4   unidenti-                                                                             pento or                                                                             amobarbi-                                                                            22    7.1                                               fied barb                                                                             amobarbi-                                                                            tal (2.5)                                                      methadone                                                                             tal                                                               5   pentobarbi-                                                                           amo, pento                                                                           pentobarbi-                                                                          31    >100                                              tal     or butabar-                                                                          tal (6.5)                                                      methadone                                                                             bital  secobarbi-                                                                    tal                                                        6*  phenobarbi-                                                                           phenobarbi-                                                                          phenobarbi-                                                                          8     1.1                                               tal     tal    tal (12)                                                       methadone                                                                 7*  phenobarbi-                                                                           phenobar-                                                                            phenobarbi-                                                                          9     0.8                                               tal     bital  tal (14)                                                       methadone                                                                 8   phenobarbi-                                                                           phenobarbi-                                                                          phenobarbi-                                                    tal     tal    tal (4)                                                                              2.9   2.2                                               morphine                                                                      methadone                                                                 9   phenobarbi-                                                                           pheno, amo,                                                                          phenobarbi-                                                                          140   64                                                tal     buta or                                                                              tal (32)                                                               pento-                                                                        barbital                                                          10  pentobarbi-                                                                           amo, buta,                                                                           amobarbi-                                                                            24    32.5                                              tal     or pheno-                                                                            tal (<.2)                                                      phenobarbi-                                                                           barbital                                                                             phenobarbi-                                                    tal            tal (12)                                                       unident.                                                                      narc.                                                                     11  phenobarbi-                                                                           pheno- phenobar-                                                                            2.4   1.1                                               tal     barbi- bital                                                          methadone                                                                             tal    (0.7)                                                      12  secobarbi-                                                                            pento- phenobar-                                                                            11    5.9                                               tal     barbi- bital                                                          methadone                                                                             tal    (0.3)                                                                         secobar-                                                                      bital (1.7)                                                13  phenobar-                                                                             pheno- phenobar-                                                                            3.7   3.0                                               bital   barbi- bital                                                          methadone                                                                             tal    (1.5)                                                          ampheta-       amobar-                                                        mine           bital                                                                         (<.2)                                                      14  phenobar-                                                                             pheno- phenobar-                                                                            2.2   1.2                                               bital   barbi- bital                                                          methadone                                                                             tal    (2.5)                                                      15  phenobar-                                                                             pheno- phenobar-                                                                            3.7   1.7                                               bital   barbi- bital                                                          methadone                                                                             tal    (1.8)                                                      16  pentobar-                                                                             amobar-                                                                              amobar-                                                                              23.0  40.0                                              bital   bital  bital                                                          methadone                                                                             butabar-                                                                             (1)                                                                    bital                                                             17  pentobar-                                                                             amobar-                                                                              amobar-                                                                              14.0  8.8                                               bital   bital  bital                                                          methadone                                                                             pento- (1.8)                                                                  barbi- secobar-                                                               tal    bital                                                                         (0.9)                                                      18  unidenti-                                                                             pheno- phenobar-                                                                            1.9   1.55                                              fied barb                                                                             barbi- bital                                                          methadone                                                                             tal    (1.7)                                                      19  phenobar-                                                                             pheno- phenobar-                                                                            17.0  5.3                                               bital   bar-   bital                                                          methadone                                                                             bital  (14.0)                                                                        pento-                                                                        barbi-                                                                        tal (<.4)                                                  20  phenobar-                                                                             pheno- phenobar-                                                                            9.4   2.4                                               bital   barbi- bital                                                          methadone                                                                             tal    (0.7)                                                      21  pentobar-                                                                             pento- pentobar-                                                                            0.5   negative                                          bital   barbi- bital  (negative)                                              methadone                                                                             tal    (0.1)                                                      __________________________________________________________________________     *These samples were all basic (pH 8 - 9.5) after standing at room             temperature with no preservative for up to two months. The enzyme assay       barbiturate levels were obtained after the pH of these samples was            adjusted to pH ≃6.0.                                       

The results show the excellent qualitative and quantitative correlationbetween the various methods. The combined enzyme assay is sensitive to0.5 μg/ml of secobarbital with somewhat less sensitivity to otherbarbiturates. As expected, the enzyme assay will be most sensitive forthe barbiturates to which the antibodies were prepared.

In accordance with the invention, concentrations required for assayingof a wide variety of ligands are of the order of 10⁻⁷ M or less withsamples of 50 μl or less of unknown. With extremely small amounts ofreagents, a very high degree of sensitivity is obtained. Furthermore,the excellent specificity of the receptor sites to a particular compoundor its close analogs permits a wide range of assay possibilities with ahigh degree of sensitivity and specificity to particular compounds.Therefore, extremely minor amounts of biologically active materials maybe assayed in the various body fluids, such as blood, saliva or urine.

The subject invention provides an extraordinarily sensitive probe forthe assaying of extremely minute amounts of specific materials with ahigh degree of specificity and accuracy. Alternatively, the method canbe used qualitatively to determine the presence or absence of particularmaterials with a high degree of specificity.

Much technology for enzyme assays has already been developed. Enzymeassays are well known: the optimum conditions for the assay, thesubstrates, and methods for detecting enzymatic activity are amplydeveloped in the literature. Furthermore, much of the work involved inradioimmunoassay is directly applicable to the subject invention. Theantisera available for radioimmunoassay are substantially applicable toligands employed in the subject invention.

Methods for bonding compounds to enzymes at other than the active siteare also well developed. There is ample literature on thefunctionalities which can be employed in bonding a particular compoundto a particular site or amino acid in an enzyme, without substantiallyaffecting the activity of the enzyme. The above examples demonstratethat the presence of an antibody when bound to a ligand which is boundto an enzyme can significantly reduce the activity of the enzyme. Thisis done either sterically or by altering the conformation of the enzyme.Furthermore, the enzymatic activity is substantially regenerated byintroducing a ligand into the medium which can effectively displace theligand bound to the enzyme, thus freeing the enzyme from the antibody.

By having an enzyme bound to a ligand, for each ligand that displaces anenzyme-bound-ligand from its receptor, a large number of substratemolecules will react and the concentration of the remaining substrate orthe product can be measured. Thus, a significant amplification results(by coupling the enzyme to a ligand) because many molecules are modifiedby virtue of the presence of a single molecule.

The subject invention permits assays of compounds which are present inextremely low concentrations or absolute amounts. First, becausereceptors are available having high specificity, one or a group ofcompounds can be determined without significant interference from othercompounds. By virtue of having one or more enzymes present in relationto a specific ligand, one can obtain a large change in concentration ofthe enzyme substrate based on a single ligand. In addition, the use ofenzymes provides a great versatility in the detection system which isemployed.

Although the foregoing invention has been described in some detail byway of illustration and example by purposes of clarity of understanding,it will be obvious that certain changes and modifications may bepracticed within the scope of the invention, as limited only by thescope of the appended claims.

What is claimed is:
 1. An enzyme-bound-ligand of the formula:##STR61##wherein: any one of the W groups other than W⁴⁴ and W⁴⁵ can be-X* or an H of any of the W groups other than W⁴⁴ or W⁴⁵ may be replacedby -X*, wherein -X* is a bond or a linking group;A* is an enzyme bondedat other than its reactive site, having n ligands, wherein n is in therange of 1 to the molecular weight of -A* divided by 2,000; W⁴⁰ and W⁴¹are hydrogen or alkyl of from 1 to 3 carbon atoms; W⁴² is hydrogen,alkyl of from 1 to 3 carbon atoms, or may be taken together with W⁴⁰ toform a ring having 6 annular members with a nitrogen as the onlyheteroatom; W⁴³ is hydrogen, hydroxyl, carbomethoxy, or may be takentogether with W⁴⁰ to form a morpholine ring, with the proviso that W⁴³is carbomethoxy, when W⁴⁰ and W⁴² are taken together; and W⁴⁴ and W⁴⁵are hydrogen, hydroxyl, or alkoxyl of from 1 to 3 carbon atoms.
 2. Anenzyme-bound-ligand of the formula: ##STR62##wherein: any one of the Wgroups other than W^(46') can be -X* or an H of any of the W groupsother than W^(46') may be replaced by -X*, wherein -X* is a bond or alinking group;A* is an enzyme bonded at other than its reactive site,having n ligands wherein n is in the range of 1 to the molecular weightof A* divided by 2,000; W^(40') and W^(41') are hydrogen or alkyl offrom 1 to 3 carbon atoms; W^(42') is hydrogen, methyl, or may be takentogether with W^(40') to form a piperidine ring; W^(43') is hydrogen,hydroxyl, or carbomethoxy with the proviso that W^(43') is carbomethoxywhen W^(42') is taken together with W^(40') ; and W^(46') is hydrogen.3. An enzyme-bound-ligand of the formula: ##STR63##wherein: one ofW^(40"'), and W^(42"') is -X**;when other than -X**, W^(40"') ishydrogen; W^(42"') is methyl; and W^(44"') is hydrogen; W^(41"') ishydrogen or methyl; X** is -Z-C═O wherein Z is hydrocarbylene of from 1to 7 carbon atoms, with the proviso that when W^(44"') is -X**, -X** is##STR64##and A** is an enzyme having n' ligands, wherein n' is in therange of 1 to 20, and said enzyme has a molecular weight in the range of10,000 to 300,000.
 4. An enzyme-bound-ligand according to claim 3,wherein said enzyme is an oxidoreductase.
 5. An enzyme-bound-ligandaccording to claim 3, wherein said enzyme is a hydrolase.
 6. Anenzyme-bound-ligand according to claim 3, which isN-carboxymethylamphetamine conjugate to lysozyme, having from 2 to 4 ofsaid amphetamine groups.
 7. An enzyme-bound-ligand according to claim 3,which is N-carboxymethylamphetamine conjugate to malate dehydrogenase,having from 2 to 22 of said amphetamine groups.
 8. Anenzyme-bound-ligand according to claim 3, which isN-carboxymethylamphetamine conjugate to glucose 6-phosphatedehydrogenase, having from 2 to 22 of said amphetamine groups.